Antibodies with enhanced or suppressed effector function

ABSTRACT

Rationally designed antibodies and polypeptides that comprise multiple Fc region amino acid substitutions that synergistically provide enhanced selectivity and binding affinity to a target Fc receptor are provided. The polypeptides are mutated at multiple positions to make them more effective when incorporated in antibody therapeutics than those having wild-type Fc components.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application No.61/436,584, filed Jan. 26, 2011, and U.S. Provisional Application No.61/318,583, filed Mar. 29, 2010. The contents of both provisionalapplications are incorporated in their entirety by reference herein.

FIELD OF INVENTION

The present invention relates to antibodies, fusion proteins andpolypeptides. Specifically, the instant invention relates to designedantibodies and polypeptides that comprise Fc region amino acidsubstitutions that synergistically provide enhanced selectivity andbinding affinity to a target Fc receptor.

BACKGROUND OF THE INVENTION

Antibodies are proteins which exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FRs). The variable domains of native heavyand light chains each comprise four FRs, largely adopting a .beta.-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the .beta.-sheet structure. The CDRs ineach chain are held together in close proximity by the FRs and, with theCDRs from the other chain, contribute to the formation of the antigenbinding site of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement; and human IgG1 and IgG3 mediateADCC more effectively than IgG2 and IgG4

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. The crystal structure of the human IgG Fc regionhas been determined (Deisenhofer, Biochemistry 20:2361-2370 (1981)). Inhuman IgG molecules, the Fc region is generated by papain cleavageN-terminal to Cys 226. The Fc region is central to the effectorfunctions of antibodies.

The effector functions mediated by the antibody Fc region can be dividedinto two categories: (1) effector functions that operate after thebinding of antibody to an antigen (these functions involve theparticipation of the complement cascade or Fc receptor (FcR)-bearingcells); and (2) effector functions that operate independently of antigenbinding (these functions confer persistence in the circulation and theability to be transferred across cellular barriers by transcytosis).Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).

While binding of an antibody to the requisite antigen has a neutralizingeffect that might prevent the binding of a foreign antigen to itsendogenous target (e.g. receptor or ligand), binding alone may notremove the foreign antigen. To be efficient in removing and/ordestructing foreign antigens, an antibody should be endowed with bothhigh affinity binding to its antigen, and efficient effector functions.

Fc Receptor (FcR)

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system effects a variety of responses, includingantibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) (reviewed in Daeron, Annu. Rev. Immunol.15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995);as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).

Several antibody effector functions are mediated by Fc receptors (FcRs),which bind the Fc region of an antibody. FcRs are defined by theirspecificity for immunoglobulin isotypes; Fc receptors for IgG antibodiesare referred to as FcγR, for IgE as Fc.epsilon.R, for IgA as Fc.alpha.Rand so on. Three subclasses of FcγR have been identified in humans:FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each FcγRsubclass is encoded by two or three genes, and alternative RNA spicingleads to multiple transcripts, a broad diversity in FcγR isoformsexists. The three genes encoding the FcγRI subclass (FcγRIA, FcγRIB andFcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1;the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) andthe two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clusteredin region 1q22. These different FcR subtypes are expressed on differentcell types (reviewed in Ravetch and Bollard, Annu. Rev. Immunol.19:275-290 (2001). For example, in humans, FcγRIIIB is found only onneutrophils, whereas FcγRIIIA is found on macrophages, monocytes,natural killer (NK) cells, and a subpopulation of T-cells. Notably,FcγRIIIA is the only FcR present on NK cells, one of the cell typesimplicated in ADCC.

FcγRI, FcγRII and FcγRIII are immunoglobulin superfamily (IgSF)receptors; FcγRI has three IgSF domains in its extracellular domain,while FcγRII and FcγRIII have only two IgSF domains in theirextracellular domains.

Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn isstructurally similar to major histocompatibility complex (MHC) andconsists of an .alpha.-chain noncovalently bound to.beta.2-microglobulin.

FcγRII (CD32), has several isoforms, IIa, IIb1, Iib2, IIb3 and IIc, andis the most widely distributed human FcγR type, being expressed on mosttypes of blood leukocytes, dendritic cells and platelets. FcγRII is alow affinity receptor that only binds to aggregated IgG. It is the onlyFcγR class to be able to bind to IgG2. The FcγRIIa is expressed on arange of cell types, including monocytes, macrophages, neutrophils,eosinophils and basophils, which it can co-activate in combination withother immunoglobulin receptors through its ITAM motifs. FcγRIIa bindsIgG antibodies attached to cells and causes lysis of those cells. Thisprocess is called antibody-dependant cell mediated cytotoxicity. TheFcγRIIb is also widely expressed but bears an immunoreceptortyrosine-based inhibitory motif (ITIM) which is necessary for itsinhibitory effects. FcγRIIb can suppress activation of B cells byinvoking negative signaling when it is cross-linked to surfaceimmunoglobulin via Ab-Ag complexes. The activation of macrophages andmonocytes via FcγR is suppressed by co-ligation of FcγRIIb. (Armour etal. (2003) “Differential binding to human FcγRIIa and FcγRIIb receptorsby human IgG wildtype and mutant antibodies,” Mol. Immunol.,40:585-593). Cells transfected to express both FcγRIIa and FcγRIIb showreduced phagocytosis relative to cells bearing FcγRIIa alone and thecytotoxicity of anti-tumour Ab was enhanced in FcγRIIb-deficient mice.(Hunter et al. (1998) “Inhibition of Fcγ receptor mediated phagocytosisby nonphagocytic Fcγ receptor.” Blood, 91:1762-1768; and Clynes et al.(2000) “Inhibitory Fc receptors modulate in vivo cytotoxicity againsttumour targets.” Nat. Med. 6:443-446). Thus, shifting relative bindingaffinity of FcγR from FcγRIIa to FcγRIIb, for example would suppressactivation of B cells and or macrophages and monocytes and would beuseful to dampen immune responses in inflammatory disorders such invarious autoimmune diseases. Conversely, shifting the relative bindingaffinity of Fcγ from FcγRIIb to FcγRIIa would lead to an enhancement ofADCC, and would be useful to enhance tumor cell killing, for example inthe treatment of cancer.

FcγRIII (CD16) has two isoforms which are able to bind to IgG1 and IgG3.The FcγRIIIa has an intermediate affinity for IgG and is expressed onmacrophages, monocytes, NK cells and subsets of T cells. FcγRIIIb is alow-affinity receptor which is selectively expressed on neutrophils.FcγRIIIa, like FCγRIIa, binds to IgG antibodies attached to cells andcauses the lysis of those cells by ADCC. FcγRIIIa binds clustered IgGmolecules bound to cell surfaces and does not bind to monomeric IgG.Therefore, ADCC occurs only when the target cell is coated withantibody. Engagement of FcγRIIIa by antibody-coated target cellsactivates NK cells to synthesize and secrete cytokines such as IFN-γ, aswell as discharge the contents of their granules, which mediate thecytolytic functions of this cell type.

Altering the effector activity of antibodies by shifting effectorfunction from otherwise inhibitory immune response to inducing ADCC andvice versa is desirable for bettering treatment outcomes in a variety ofdiseases and conditions. Lazar et al. (2006) Proc. Natl. Acad. Sci.U.S.A., 103:4005, Stavenhagen et al. (2007) Cancer Res. 67:8882,Oganesyan et al. (2008) Mol. Immunol. 45:1872, Veri et al. (2007)Immunology, 121:392, and Shields, et al. (2001) J. Biol. Chem. 276:6591disclose efforts in this research area.

Presta et al. (U.S. Pat. No. 6,737,056) discloses polypeptidescomprising a variant Fc region. The disclosed variations are based onsingle position mutations. By performing alanine scans, Presta et al.discloses single position Fc region amino acid modification at positions238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335,338, 373, 376, 414, 416, 419, 435, 438 or 439; these single positionmutations are purported to result in reduced binding to FcγRII. Prestaet al. also discloses that systematic alanine scans of the entire Fcregion purportedly show that an Fc region amino acid modification at anyone of amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269,270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338,340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 results in reducedbinding to FcγRIII. Presta et al. also discloses polypeptides withincreased binding to an FcγRII comprising single position amino acidmodifications at any one of amino acid positions 255, 256, 258, 267,268, 272, 276, 280, 283, 285, 286, 290, 301, 305, 307, 309, 312, 315,320, 322, 326, 330, 331, 337, 340, 378, 398 or 430 of the Fc regionidentified by alanine scans as well. Presta et al. discloses one singleinstance where two Fc amino acid positions are mutated at the same time(i.e., modifications S317A and K353A), however Presta et al. does notsuggest that these double mutations provide any synergistic effect inobtaining a desired binding profile of the modified polypeptide toFcγRII. Presta et al. is completely silent on potential synergism ofsimultaneous modifications at more than a single amino acid position.

Lazar et al. (U.S. Pat. No. 7,317,091) discloses antibodies comprisingan amino acid modification at position 332 in the Fc region purportedlyresulting in altered binding to an FcγR. Lazar et al. discloses thatindividual substitutions in positions 234, 235, 239, 240, 243, 264, 266,272, 274, 278, 325, 328, 330, and 332 purportedly effect the binding toan FcγR. While Lazar al. purports to disclose synergy of Fc variantswhen combined with engineered glycoforms, Lazar et al. is silent onpotential synergism that may be provided by a selection ofsimultaneously modified Fc amino acid positions, regardless ofadditional synergism that may be provided by engineered glycoforms.

Stavenhagen (U.S. Pat. No. 7,632,497) discloses molecules having avariant Fc region, wherein the variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region. Thesemodified molecules purportedly confer an effector function to amolecule, where the parent molecule does not detectably exhibit thiseffector function.

Current approaches to optimize the Fc region in therapeutic monoclonalantibodies and soluble polypeptides fused to Fc regions have focused ona limited number of single amino acid changes based on alanine screens,site-directed mutagenesis etc. Other approaches in engineering Fcregions have focused on the glycosylation of the Fc region to optimizeFc region function. Still other approaches have focused on Femodifications that purportedly confer effector function by modifyingwild type molecules that lack effector function, but do not purport toincrease or otherwise modify existing effector function of a wild typemolecule.

There is currently no known Fc protein or polypeptide that is optimizedto bind a particular FcγR of interest with very high specificity ascompared to other Fcγ receptors. Although the effect of individual Feregion amino acid mutations on the binding with certain Fcγ receptors iswell understood, previous studies fail to describe the effect ofsimultaneous changes to multiple amino acids. Additionally, the priorart does not suggest suitable replacements for substituted Fc regionamino acids to obtain optimal binding to the FcγR of interest, includingmodification of effector function of a wild type molecule that alreadyhas detectable effector function.

Hence, there is a need in the art for polypeptides and antibodies thatcomprise multiple, synergistic amino acid substitutions in the Fc regionsuch that the antibody or fusion protein is optimized for binding to theFcγR isoform of choice.

SUMMARY OF THE INVENTION

The instant compositions provide polypeptides, fusion protein andantibodies comprising Fc region that bind a target FcγR with very highspecificity. These polypeptides and antibodies are designed by rationalanalysis of the binding of each FcγR with the Fc region and bysubsequent design of multiple (three or more) amino acid substitutionsthat synergistically provide desired selectivity and binding affinityfor the target Fc receptor. Accordingly, the polypeptides and proteinsprovided herein comprise multiple variations in the Fc region ascompared to the wild type Fc region, said variations tailored toimproving the specificity for the FcγR under consideration, and forobtaining a selected binding profile based on enthalpic and entropicfactors optimized by the choice of the most favorable amino acid foreach position.

One embodiment provides a polypeptide comprising a variant Fc region,wherein said variant Fc region comprises three or more amino acidmodifications relative to a wild-type Fc region, and has an alteredeffect relative to a polypeptide comprising a wild-type Fc region;wherein at least two of the three or more modifications provide asynergistic effect compared to single position modifications at the atleast two positions thereby exhibiting a selected binding profile to Fcγreceptors.

Another embodiment provides a polypeptide wherein the amino acidmodifications produce amino acid interactions and dynamics that resultin enhanced binding free energy to a first Fcγ receptor whilediminishing binding affinity to a second Fcγ receptor compared to apolypeptide that lacks the at least three or more amino acidmodifications. The first Fcγ receptor may be FcγRIIIa receptor and thesecond Fcγ receptor may be FcγRIIa or FcγRIIb.

Another embodiment provides a polypeptide wherein the amino acidmodifications produce favorable FcγRIIIa-specific interactions and/orunfavorable interactions with FcγRIIa and/or FcγRIIb receptors.

Another embodiment provides a polypeptide wherein the amino acidmodifications have minimal impact on the FcγRIIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIaand/or FcγRIIb.

One embodiment provides a polypeptide wherein the first Fcγ receptor isFcγRIIa receptor and the second Fcγ receptor is FcγRIIIa or FcγRIIb.

One embodiment provides a polypeptide wherein the amino acidmodifications produce favorable FcγRIIa-specific interactions and/orunfavorable interactions with FcγRIIIa and/or FcγRIIb receptors.

One embodiment provides a polypeptide wherein the amino acidmodifications have minimal impact on the FcγRIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIIaand/or FcγRIIb.

One embodiment provides a polypeptide wherein the Fcγ receptor withenhanced binding free energy is FcγRIIb receptor and the Fcγ receptorwith diminished binding affinity is FcγRIIIa receptor or FcγRIIareceptor.

One embodiment provides a polypeptide wherein the amino acidmodifications produce favorable FcγRIIb-specific interactions and/orunfavorable interactions with FcγRIIIa and/or FcγRIIa receptors.

One embodiment provides a polypeptide wherein the amino acidmodifications have minimal impact on the FcγRIIb receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIIaand/or FcγRIIa.

One embodiment provides a polypeptide wherein the amino acidsubstitutions reduce or disrupt the binding affinity to the FcγRIIareceptor, the FcγRIIIa receptor and the FcγRIIb receptor when comparedto the wild-type polypeptide.

One embodiment provides a polypeptide wherein binding of the polypeptidecomprising a wild-type Fc region to Fcγ receptors is detectable by an invitro assay.

In certain embodiments of the instant invention, are methods forengineering optimized Fc polypeptides and proteins. It is an object ofthe present disclosure to provide design strategies that may be used toguide Fe optimization. It is a further object of the present disclosureto provide computational screening methods that may be used to design Fcproteins. It is a further object of the present disclosure to providemethods for generating libraries for experimental testing. It is afurther object of the present disclosure to provide experimentalproduction and screening methods for obtaining optimized Fe proteins.

In certain embodiments are provided isolated nucleic acids encoding theFc proteins described herein. In certain embodiments are providedvectors comprising said nucleic acids, optionally, operably linked tocontrol sequences. In certain embodiments are provided host cellscontaining the vectors, and methods for producing and optionallyrecovering the Fc proteins.

In certain embodiments are provided antibodies and polypeptides thatcomprise the Fc proteins disclosed herein. Said antibodies andpolypeptides may find use in a therapeutic product.

The present disclosure provides compositions comprising antibodies andpolypeptides that comprise the Fc proteins described herein, and aphysiologically or pharmaceutically acceptable carrier or diluent.

In certain embodiments are provided therapeutic and diagnostic uses forantibodies and polypeptides that comprise the Fc proteins disclosedherein.

Provided herein are polypeptides comprising a variant Fc region, whereinsaid variant Fc region comprises at least three amino acid modificationsrelative to a wild-type Fc region, and has an altered effect relative toa polypeptide comprising a wild-type Fc region or variant Fc regioncomprising only one or two amino acid modifications; and wherein atleast two of the modifications provide a synergistic effect compared tosingle position modifications thereby exhibiting a selected bindingprofile to Fcγ receptors. In certain embodiments, one or both amino acidmodifications are located between positions 234-330 according to the EUindex. In certain embodiments, the modifications do not comprisesimultaneous substitution at positions 317 and 353 according to the EUindex. In some embodiments, the amino acid modifications do not comprisea substitution at position 332 according to the EU index. In certainembodiments, the polypeptides comprise the modificationsL235A/S239E/D265E. In some embodiments, the polypeptides comprise themodifications A327H/E269L/K236A. In a certain embodiments, thepolypeptides comprise the modifications G237F/D270Q/S239E. In some otherembodiments, the polypeptides comprise the modificationsA330V/I332L/K326. In one embodiment, the polypeptides comprise themodifications G236S/A327H/A330I.

In certain embodiments of the polypeptides described herein, the aminoacid modifications produce amino acid interactions and dynamics thatresult in enhanced binding affinity and/or specificity to a first Fcγreceptor while diminishing binding affinity and/or specificity to asecond Fcγ receptor compared to a polypeptide that lacks the three ormore amino acid modifications. In some of these embodiments, the firstFcγ receptor is FcγRIIIa receptor and the second Fcγ receptor is FcγRIIaor FcγRIIb. In certain embodiments, the amino acid modifications producefavorable FcγRIIIa-specific interactions and/or unfavorable interactionswith FcγRIIa and/or FcγRIIb receptors. In some embodiments, the aminoacid modifications have minimal impact on the FcγRIIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIaand/or FcγRIIb.

Provided herein are polypeptides comprising a variant Fc region, whereinsaid variant Fc region comprises at least three amino acid modificationsrelative to a wild-type Fc region, wherein the amino acid modificationsproduce amino acid interactions and dynamics that result in enhancedbinding affinity and/or specificity to a FcγRIIIa receptor whilediminishing binding affinity and/or specificity to FcγRIIa or FcγRIIbreceptor compared to a polypeptide that lacks the two amino acidmodifications. In some embodiments, the polypeptide comprisesmodifications S239E/D265S/I332E. In certain embodiments, the polypeptidecomprises the modifications G237F/S239E/A327H. In certain otherembodiments, the polypeptide comprises modificationsH268D/E269L/S298A/K326A/A327H. In some embodiments, the polypeptidecomprises the modifications L235A/S239E/D265E/A327H. In an embodiment,the polypeptide comprises the modifications G237F/S239E/D270N. In someembodiments, the polypeptide comprises the modificationsG236E/G237F/S239E. In an embodiment, the polypeptide comprises themodifications S239E/D265SI332E and alternatively H268D. In certainembodiments, the polypeptide comprises modifications selected from thegroup of G237F/S239E/D265E, S239E/S298A/K326A/A327H, andG236E/D270N/A327V/I332E. In certain embodiments, the polypeptidecomprises the modifications S298A/K326A/A327H wherein the polypeptidehas improved binding selectivity to FcγRIIIa receptor as compared to apolypeptide lacking the S298A/K326A/A327H modifications.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein the aminoacid modifications produce amino acid interactions and dynamics thatresult in enhanced binding affinity and/or specificity to a FcγRIIareceptor while diminishing binding affinity and/or specificity toFcγRIIIa or FcγRIIb receptor compared to a polypeptide that lacks atleast one of the amino acid modifications. In some embodiments, thepolypeptide comprises modifications G237F, A327L and A330I. In certainembodiments, the amino acid modifications produce favorableFcγRIIa-specific interactions and/or unfavorable interactions withFcγRIIIa and/or FcγRIIb receptors. In certain embodiments, the aminoacid modifications have minimal impact on the FcγRIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIIaand/or FcγRIIb. In certain embodiments, the polypeptide comprisesmodifications G237F, S239E and H268D. In some embodiments, thepolypeptide comprises modifications D265E/S267D/A330S.

In a further aspect, provided herein are polypeptides comprising avariant Fc region, wherein said variant Fc region comprises at leastthree amino acid modifications relative to a wild-type Fc region,wherein the amino acid modifications produce amino acid interactions anddynamics that result in enhanced binding affinity and/or specificity toa FcγRIIb receptor while diminishing binding affinity and/or specificityto FcγRIIIa or FcγRIIa receptor compared to a polypeptide that lacks atleast one of the amino acid modifications. In certain embodiments, theamino acid modifications produce favorable FcγRIIb-specific interactionsand/or unfavorable interactions with FcγRIIIa and/or FcγRIIa receptors.In certain embodiments, the amino acid modifications have minimal impacton the FcγRIIb receptor while producing detrimental effects on bindingof the polypeptide to FcγRIIIa and/or FcγRIIa. In certain embodiments,the polypeptide comprises modifications S239D/D265S/S298A/I332E. In someother embodiments, the polypeptide comprises the modificationsG237F/S298A/A330L/I332E. In some embodiments the polypeptide comprisesthe modifications H268D, K326A, A327H and alternatively one or both ofE269L and S298A. In an embodiment, the polypeptide comprises themodifications G237F/V266L/S267D. In some embodiments, the polypeptidescomprise the modifications L234F/S267G/N325L or L234F/S267E/N325L. In anembodiment, the polypeptide comprises modificationsG236A/S239D/D270L/I332E.

In certain aspects described herein, the binding of the polypeptidecomprising a wild-type Fc region to Fcγ receptors is detectable by an invitro assay.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein one of themodifications comprises the mutation S239E wherein the polypeptide hashigher selectivity in binding to the FcγRIIIa receptor compared to apolypeptide that lacks the S239E mutation.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein one of themodifications comprises the mutation S239E one of the modificationscomprises the mutation S298A wherein the polypeptide has reduced bindingaffinity to FcγRIIa and FcγRIIb receptors compared to a polypeptide thatlacks the S298A mutation.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, said modificationsselected from D270L/Y300L/A330K, G237F/S267G/N325F, G237F/V266L/S267D,L234F/S267G/N325L, L234F/S267E/N325L, G237F/S239E/A327H,G237F/A327L/A330I, S239E/A327L/A330I, S239E/S267E/H268D,G237F/S239E/D270N, G236E/G237F/S239E, S239E/D265S/I332E,G237F/S239E/D265E, G237F/S239E/H268D, H268E/D270E/S267G,H268D/K326A/A327H, D265E/S267D/A330S, L235A/S239E/D265E,A327H/E269L/K236A, G237F/D270Q/S239E, A330V/I332L/K326, andG236S/A327H/A330I.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least four aminoacid modifications relative to a wild-type Fc region, said modificationsselected from L235A/S239E/D265E/A327H, S239E/D265S/H268D/I332E,S239D/D265S/S298A/1332E, S239E/S298A/K326A/A327H,G237F/S298A/A330L/I332E, and G236E/D270N/A327V/I332E,G236A/S239D/D270L/I332E and H268D/E269L/S298A/K326A/A327H.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein the atleast three amino acid modifications are selected from the groupconsisting of: L234Q, L234N, L235A, G236E, E236L, E236D, G237F, G237N,S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268D, H268E, E269L,E269L, D270N, D270I, D270E, S298A, K326A, K326D, A327H, A327V, A327L,A327T, A330V, A330L, A330W, A330I, A330S, I332L, I332D, and 1332E.

In an aspect provided herein are polypeptides comprising a variant Fcregion, said variant Fc region comprises a combination of amino acidmodifications wherein said combination is selected from the groupconsisting of: L235A/S239E/D265E; L235A/G237F/D265E; S239E/E269D/A327H;S239E/G237N/A327H; S239E/G237F/A327V; G237F/D270I/S239E;G237F/A327L/S239E; A327H/E269L/K326A; A330V/I332L/S239E;A327T/E269L/K326A; D270N/A327T/K326A; A330V/I332L/S239E;A330W/I332D/S239E; G236E/D265E/A327H/A330I; D270N/S298A/A327V;G236E/D265E/D270N/A327H/A330I; G236E/D270N/A327H/A330I;G236E/D270N/A327V/I332E; G236E/D270N/A327V/G237F;L234N/S239E/A330I/I332E; L234Q/S239E/A330I/I332E;L234Q/S239E/A330I/I332E/S298A; G237F/S239D/D265E/D270N/S298A;G237F/S239E/D270N/A330L/I332E; G237F/S239E/D270N/A330L/I332E/S298A;S239E/G237F/A327H; G237F/A327L/A330I; S239E/A330I/A327L;D265E/S239E/L235A/A327H; S267E/S239E/H268D; G237F/D270N/S239E;S239E/G237F/G236E; 1332E/D265S/S239E/H268D; I332E/D265S/S239E;D265E/S239E/G237F; S239E/H268D/G237F; S298A/D265S/S239D/1332E;S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E; H268E/D270E/S267G;H268D/K326A/A327H; H268D/K326A/A327H/E269L/S298A; A330S/D265E/S267D;S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G

In an aspect, provided herein are polypeptides that comprise a variantFc region, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region wherein when saidvariant Fc region comprises amino acid modification H268D, said variantdoes not comprise the modification S267E.

In certain embodiments of the polypeptides described herein, the Fcregion of the parent polypeptide is a human IgG Fc region. In some ofthese embodiments, the human IgG Fc region is a human IgG1, IgG2, IgG3,or IgG4 Fc region.

In certain embodiments of the polypeptides described herein, thepolypeptide is an antibody. In some embodiments, the antibody is amonoclonal antibody, a humanized antibody, or a human antibody.

In an aspect is a nucleic acid comprising: a nucleotide sequenceencoding a polypeptide described herein. In certain embodiments is avector, comprising the nucleic acid.

In an aspect is a method for producing a polypeptide or proteindescribed herein, said method comprising: (i) culturing in a medium ahost cell comprising a nucleic acid encoding said polypeptide, underconditions suitable for the expression of said polypeptide; and (ii)recovering the polypeptide from said medium.

In an aspect described herein is a therapeutic antibody thatspecifically binds a cancer target antigen, said therapeutic antibodycomprising a variant Fc region polypeptide described herein. In certainembodiments, the therapeutic antibody is selected from the groupconsisting of abagovomab, adalimumab, alemtuzumab, aurograb,bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab,canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab,denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab,ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab,motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab,ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab,ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab,tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab,zalutumumab, and any other antibodies. In certain embodiments, thetarget antigen is selected from the group consisting of a-chain (CD25)of IL-2R, Amyloid beta, anti-EpCAM×anti-CD3, BLyS (or BAFF), CD11a,CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F proteinof RSV, G250, glycoprotein IIb/IIIa R, HER2, HER2/neu R, Hsp90, IgEantibody, IL-12/IL-23, IL-1b, IL-5, IL-6 receptor, Integrinalpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF alpha, VEGF-A, and othertherapeutically advantageous targets.

In an aspect described herein is a method of treating cancer in apatient having a cancer characterized by a cancer antigen, said methodcomprising administering to said patient a therapeutically effectiveamount of a therapeutic antibody described herein. In certainembodiments, the patient is human.

In an aspect described herein is a method of treating immune disordersin a patient having an immune disorder characterized by an immuneantigen, said method comprising administering to said patient atherapeutically effective polypeptide, antibody or protein describedherein.

In an aspect is a pharmaceutical composition, said compositioncomprising a therapeutically effective amount of a polypeptide describedherein, and a pharmaceutically acceptable carrier.

In an aspect described herein are polypeptides, said polypeptidescomprising a variant Fc region with at least three amino acidsubstitutions, and wherein said polypeptides are more effective atmediating antibody-dependent cellular cytotoxicity (ADCC) relative towild type. In certain embodiments, the polypeptide comprising a variantFc region is about 1.5 to about 100 fold more effective in mediatingADCC relative to wild type. In certain embodiments, the polypeptidecomprising a variant Fc region is about 2 to about 50 fold moreeffective in mediating ADCC relative to wild type.

In an aspect described herein are polypeptides, said polypeptidescomprising a variant Fc region with at least three amino acidsubstitutions, wherein the polypeptide is more effective at mediatinginhibition of inflammatory immune responses relative to wild type. Incertain embodiments, the polypeptide comprising a variant Fc region isabout 2 fold more effective in mediating inhibition of inflammatoryimmune responses relative to wild type. In some embodiments, thepolypeptide comprising a variant Fc region is about 10 fold moreeffective in mediating inhibition of inflammatory immune responsesrelative to wild type. In certain embodiments, the polypeptidecomprising a variant Fc region is about 50 fold more effective inmediating inhibition of inflammatory immune responses relative to wildtype. In certain embodiments, the polypeptide comprising a variant Fcregion is about 100 fold more effective in mediating inhibition ofinflammatory immune responses relative to wild type.

Also provided herein is a method for identifying Fc variant polypeptidesin silico based on calculated binding affinities to FcγRIIa, FcγRIIband/or FcγRIIIa. In certain embodiments, the method of identifying Fcvariant polypeptides in silico further calculates in silicoelectrostatics, solvation, packing, packing density, hydrogen binding,and entropic effects of said Fc variant polypeptides. In certainembodiments, the method of identifying Fc variant polypeptides in silicofurther comprises constructing the identified Fc variant polypeptidesand expressing said polypeptides in the context of an antibody inmammalian cells.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1: Effects of the A327H and S239E mutations: A portion of thebinding interface is shown between the chain-B of the antibody Fc(green) and the various Fcγ receptors (wheat). The wild-type Fc toFcγRIIIa structure is shown on top with Fc variant-induced changes instructure and dynamics for each receptor shown on the bottom. Side-chainpositions to be mutated are colored in dark green and the residue labelsare in red. Dashed lines denote electrostatic interactions with thedistances shown in angstroms (Å). For the FcγRIIa, the two displayedconformations of His134 are the maximum allowable conformationalmovements with the intermediate conformers omitted for clarity.

FIG. 2: Effects of the S239E mutation: A portion of the bindinginterface is shown between the chain-A of the antibody Fc (green) andthe receptors (wheat) FcγRIIIa (left) and FcgRIIa & IIb (right). Mutatedside-chain positions are colored in dark green with the residue labelsin red. Dashed lines denote electrostatic interactions with thedistances shown in angstroms (Å). FcγRIIa and FcγRIIb both containconserved Tyr160 and Thr161 residues and are shown together for clarity.

FIG. 3: Effects of the S298A mutation: A portion of the bindinginterface is shown between the chain-B of the antibody Fc (green) andthe various Fcγ receptors (wheat). The wild-type Fc is shown on the leftwith the variant Fc shown on the right. Interactions with the FcγRIIaand IIb receptors are shown on the top while the FcγRIIIa is shown onthe bottom. The S298A mutation is labeled in red with the dashed linesdenoting electrostatic interactions with the intermolecular distancesshown in angstroms (Å). For the FcγRIIIa system, the displayedconformations for Lys128 and Asn126 are the maximum allowableconformational movements with the intermediate conformers omitted forclarity.

FIG. 4: In vitro binding profiles to the three Fcγ receptors: The invitro binding (green) of the S239E/S298A/K326A/A327H variant (left) andthe S239E control (right) were measured using surface plasmon resonancewith binding reported as the association constant (K_(A)) in molar [M]in comparison to the wild-type Herceptin antibody (blue).

DETAILED DESCRIPTION OF THE INVENTION

Embodiments disclosed herein are drawn to polypeptides, fusion proteinand antibodies comprising at least three substitutions in the Fc regionwherein the polypeptide binds a target FcγR receptor with higherspecificity compared to a polypeptide that lacks the at least threesubstitutions. These polypeptides and antibodies are designed byrational analysis of the binding of each FcγR with the Fc region and bysubsequent design of multiple amino acid substitutions thatsynergistically provide enhanced selectivity and binding affinity forthe target Fc receptor. Accordingly, the polypeptides and proteinsprovided herein comprise multiple variations in the Fc region ascompared to the wild type Fc region, said variations tailored toimproving the specificity for the FcγR under consideration, and forobtaining maximum energetically favorable binding based on enthalpic andentropic factors optimized by the choice of the most favorable aminoacid for each substituted position.

One embodiment provides a polypeptide comprising a variant Fc region,wherein said variant Fc region comprises three or more amino acidmodifications relative to a wild-type Fc region, and has an alteredeffect relative to a polypeptide comprising a wild-type Fc region;wherein at least two of the three or more modifications provide asynergistic effect compared to single position modifications at the atleast two positions thereby exhibiting a selected binding profile to Fcγreceptors.

Another embodiment provides a polypeptide wherein the amino acidmodifications produce amino acid interactions and dynamics that resultin enhanced binding free energy to a first Fey receptor whilediminishing binding affinity to a second Fcγ receptor compared to apolypeptide that lacks the at least three or more amino acidmodifications. In certain embodiments, the first Fcγ receptor isFcγRIIIa receptor and the second Fcγ receptor is FcγRIIa or FcγRIIb.

Another embodiment provides a polypeptide wherein the amino acidmodifications produce favorable FcγRIIIa-specific electrostaticinteractions and steric repulsion to FcγRIIa and/or FcγRIIb receptors.

Another embodiment provides a polypeptide wherein the amino acidmodifications have minimal impact on the FcγRIIIa receptor whileproducing detrimental effects onbinding of the polypeptide to FcγRIIaand/or FcγRIIb.

Another embodiment provides a polypeptide of wherein the amino acidsubstitutions preserve the binding interface and the protein-proteininteractions with the FcγRIIIa when compared to the wild-type Fc, andresult in disruption of binding of the polypeptide to the FcγRIIareceptor is disrupted.

Any patent, application or other reference cited or repeated below, orabove, is incorporated by references in its entirety for all purposes aswell as for the proposition that is recited.

The following definitions may be used to understand the compositions andmethods provided herein, but are meant to encompass scientificequivalents.

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.

A “parent polypeptide” is a polypeptide comprising an amino add sequencewhich lacks one or more of the Fc region modifications disclosed hereinand which differs in effector function compared to a polypeptide variantas herein disclosed. The parent polypeptide may comprise a nativesequence Fc region or an Fc region with pre-existing amino acid sequencemodifications (such as additions, deletions and/or substitutions).

As used herein, “synergistic” means that the FcγR binding of the Fcprotein designed with multiple amino acid substitutions is greater thantheir additive binding observed for individual substitutions.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally comprises two constant domains,CH2 and CH3.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cγ2”domain) usually extends from about amino acid 231 to about amino acid340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22:161-206 (1985).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG)

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Clq binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

A “native sequence Fc region” comprises an amino add sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one“amino acid modification” as herein defined. The variant Fc region hasat least one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide. In certain embodiments, thevariant Fc region herein possesses at least about 80% homology with anative sequence Fc region and/or with an Fc region of a parentpolypeptide, and most preferably at least about 90% homology therewith,more preferably at least about 95% homology therewith.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

The term “Fc region-containing polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, in certain embodiments, the FcR is onewhich binds an IgG antibody (a gamma receptor) and includes receptors ofthe FcγRI, FcγRII, and FcγRIII subclasses, including allelic variantsand alternatively spliced forms of these receptors. FcγRII receptorsinclude FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,including those to be identified in the future are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of materal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. The primary cells for mediating ADCC, NK cells,express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

A polypeptide with “altered” FcR binding affinity or ADCC activity isone which has either enhanced or diminished FcR binding activity and/orADCC activity compared to a parent polypeptide or to a polypeptidecomprising a native sequence Fc region. The polypeptide which “displaysincreased binding” to an FcR binds at least one FcR with better affinitythan the parent polypeptide. The polypeptide which “displays decreasedbinding” to an FcR, binds at least one FcR with worse affinity than aparent polypeptide. Such variants which display decreased binding to anFcR may possess little or no appreciable binding to an FcR, e.g., 0-20%binding to the FcR compared to a native sequence IgG Fc region, e.g. asdetermined in the Examples herein.

The polypeptide which binds an FcR with “better affinity” than a parentpolypeptide, is one which binds any one or more of the above identifiedFcRs with substantially better binding affinity than the parentantibody, when the amounts of polypeptide and parent polypeptide in thebinding assay are essentially the same. For example, the polypeptidewith improved FcR binding affinity may display from about 1.15 fold toabout 100 fold, e.g. from about 1.2 fold to about 50 fold improvement inFcR binding affinity compared to the parent polypeptide, where FcRbinding affinity is determined, for example, as disclosed in theExamples herein.

The polypeptide which “mediates antibody-dependent cell-mediatedcytotoxicity (ADCC) in the presence of human effector cells moreeffectively” than a parent antibody is one which in vitro or in vivo issubstantially more effective at mediating ADCC, when the amounts ofpolypeptide variant and parent antibody used in the assay areessentially the same. Generally, such polypeptides will be identifiedusing the in vitro ADCC assay as herein disclosed, but other assays ormethods for determining ADCC activity, e.g. in an animal model etc, arecontemplated. The preferred polypeptide is from about 1.5 fold to about100 fold, e.g. from about two fold to about fifty fold, more effectiveat mediating ADCC than the parent, e.g. in the in vitro assay disclosedherein.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion. Incertain embodiments the amino acid modification herein is asubstitution.

An “amino acid modification at” a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. By insertion “adjacent” a specified residue is meantinsertion within one to two residues thereof. In certain embodiments theinsertion is N-terminal or C-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); Isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro): serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, omithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301-336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. In certainembodiments, the insertion consists of the insertion of one or two aminoacid residues. In certain other embodiments, are larger “peptideinsertions”, e.g. insertion of about three to about five or even up toabout ten amino acid residues. In these embodiments the insertedresidue(s) are naturally occurring or non-naturally occurring asdisclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S—S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. Prior to the present disclosure,FcγR binding was generally attributed to amino acid residues in thelower hinge region of an IgG Fc region.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the complement dependentcytotoxicity (CDC) pathway. Human C1q can be purchased commerciallyfrom, e.g. Quidel, San Diego, Calif.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the .alpha.chain thereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcRαchain.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

“Antibody fragments”, as defined herein, comprise a portion of an intactantibody, generally including the antigen binding or variable region ofthe intact antibody or the Fc region of an antibody which retains FcRbinding capability. Examples of antibody fragments include linearantibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. In certain embodiments, theantibody fragments retain at least part of the hinge and optionally theCH1 region of an IgG heavy chain. In some embodiments the antibodyfragments retain the entire constant region of an IgG heavy chain, andinclude an IgG light chain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal,” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. In certain embodiments the monoclonalantibodies to be used in accordance with the present disclosure are madeby the hybridoma method first described by Kohler et al., Nature 256:495(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). In some embodiments “monoclonal antibodies” are isolatedfrom phage antibody libraries using the techniques described in Clacksonet al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanImmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (ie. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with an immunoglobulinconstant domain. Structurally, the immunoadhesins comprise a fusion ofthe adhesin amino acid sequence with the desired binding specificitywhich is other than the antigen recognition and binding site (antigencombining site) of an antibody (i.e. is “heterologous”) and animmunoglobulin constant domain sequence.

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding ability of a corresponding nativereceptor. In a specific embodiment, the receptor is from a cell-surfacepolypeptide having an extracellular domain that is homologous to amember of the immunoglobulin supergenefamily. Other receptors, which arenot members of the immunoglobulin supergenefamily but are nonethelessspecifically covered by this definition, are receptors for cytokines,and in particular receptors with tyrosine kinase activity (receptortyrosine kinases), members of the hematopoietin and nerve growth factorreceptor superfamilies, and cell adhesion molecules, e.g. (E-, L- andP-) selectins.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability of a corresponding native ligand. Thisdefinition, among others, specifically includes binding sequences fromligands for the above-mentioned receptors.

An “antibody-immunoadhesin chimera” comprises a molecule that combinesat least one binding domain of an antibody (as herein defined) with atleast one immunoadhesin (as defined in this application). Exemplaryantibody-immunoadhesin chimeras are the bispecific CD4-IgG chimerasdescribed in Berg et al., PNAS (USA) 88:4723-4727 (1991) and Chamow etal., J. Immunol. 153:4268 (1994).

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinadeous solutes. In certain embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with thepolypeptide variant. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. In one embodiment, the disorder iscancer.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. In certain embodiments the label is itself detectable(e.g., radioisotope labels or fluorescent labels). In some otherembodiments, the label catalyzes chemical alteration of a substratecompound or composition which is detectable. An exemplary embodimentcomprises an enzymatic label that catalyzes a chemical alteration of asubstrate compound or composition which is detectable.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “molecular complex” when used herein refers to the relativelystable structure which forms when two or more heterologous molecules(e.g. polypeptides) bind (preferably noncovalently) to one another. Thepreferred molecular complex herein is an immune complex.

“Immune complex” refers to the relatively stable structure which formswhen at least one target molecule and at least one heterologous Fcregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody aggregates and target molecule-immunoadhesinaggregates. The term “immune complex” as used herein, unless indicatedotherwise, refers to an ex vivo complex (i.e. other than the form orsetting in which it may be found in nature). However, the immune complexmay be administered to a mammal, e.g. to evaluate clearance of theimmune complex in the mammal.

The term “target molecule” refers to a molecule, usually a polypeptide,which is capable of being bound by a heterologous molecule and has oneor more binding sites for the heterologous molecule. The term “bindingsite” refers to a region of a molecule to which another molecule canbind. The “first target molecule” herein comprises at least two distinctbinding sites (for example, two to five separate binding sites) for ananalyte (e.g. an Fc region-containing polypeptide) such that at leasttwo analyte molecules can bind to the first target molecule. In apreferred embodiment, the two or more binding sites are identical (e.g.having the same amino acid sequence, where the target molecule is apolypeptide). An “analyte” is a substance that is to be analyzed. Thepreferred analyte is an Fc region-containing polypeptide that is to beanalyzed for its ability to bind to an Fc receptor.

A “receptor” is a polypeptide capable of binding at least one ligand.The preferred receptor is a cell-surface receptor having anextracellular ligand-binding domain and, optionally, other domains (e.g.transmembrane domain, intracellular domain and/or membrane anchor). Thereceptor to be evaluated in the assay described herein may be an intactreceptor or a fragment or derivative thereof (e.g. a fusion proteincomprising the binding domain of the receptor fused to one or moreheterologous polypeptides). Moreover, the receptor to be evaluated forits binding properties may be present in a cell or isolated andoptionally coated on an assay plate or some other solid phase.

The phrase “low affinity receptor” denotes a receptor that has a weakbinding affinity for a ligand of interest, e.g. having a bindingconstant of about 50 nM or worse affinity. Exemplary low affinityreceptors include FcγRII and FcγRIII.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γγ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys. By “library” herein is meant a set of Fc proteinsin any form, including but not limited to a list of nucleic acid oramino acid sequences, a list of nucleic acid or amino acid substitutionsat variable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising the Fcprotein proteins, either in purified or unpurified form.

By “Fc-fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to an Fc region or a derivative thereof.Fc fusion is herein meant to be synonymous with the terms“immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin”(sometimes with dashes) as used in the prior art (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general can be any protein or smallmolecule. The role of the non-Fc part of an Fc fusion, i.e. the fusionpartner, is to mediate target binding, and thus it is functionallyanalogous to the variable regions of an antibody. Virtually any proteinor small molecule may be linked to Fc to generate an Fc fusion. Proteinfusion partners may include, but are not limited to, the target-bindingregion of a receptor, an adhesion molecule, a ligand, an enzyme, acytokine, a chemokine, or some other protein or protein domain. Smallmolecule fusion partners may include any therapeutic agent that directsthe Fc fusion to a therapeutic target. Such targets may be any molecule,preferrably an extracellular receptor, that is implicated in disease.Two families of surface receptors that are targets of a number ofapproved small molecule drugs are G-Protein Coupled Receptors (GPCRs),and ion channels, including K+, Na+, Ca+ channels. Nearly 70% of alldrugs currently marketed worldwide target GPCRs. Thus the Fc proteinsdescribed herein may be fused to a small molecule that targets, forexample, one or more GABA receptors, purinergic receptors, adrenergicreceptors, histaminergic receptors, opiod receptors, chemokinereceptors, glutamate receptors, nicotinic receptors, the 5HT (serotonin)receptor, and estrogen receptors. A fusion partner may be asmall-molecule mimetic of a protein that targets a therapeuticallyuseful target. Specific examples of particular drugs that may serve asFc fusion partners can be found in L. S. Goodman et at, Eds., Goodmanand Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill, NewYork, ed. 9, 1996). Fusion partners include not only small molecules andproteins that bind known targets for existing drugs, but orphanreceptors that do not yet exist as drug targets. The completion of thegenome and proteome projects are proving to be a driving force in drugdiscovery, and these projects have yielded a trove of orphan receptors.There is enormous potential to validate these new molecules as drugtargets, and develop protein and small molecule therapeutics that targetthem. Such protein and small molecule therapeutics are contemplated asFc fusion partners that employ the Fc proteins described herein. Avariety of linkers, defined and described below, may be used tocovalently link Fc to a fusion partner to generate an Fc fusion.

By “Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136). Fc ligands may includeundiscovered molecules that bind Fc.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoglobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic .quadrature.-sandwich foldingtopology. The known Ig domains in the IgG class of antibodies are V_(H),Cγ1, Cγ2, Cγ3, V_(L), and C_(L).

By “parent polypeptide” or “precursor polypeptide” (including Fc parentor precursors) as used herein is meant a polypeptide that issubsequently modified to generate a variant. Said parent polypeptide maybe a naturally occurring polypeptide, or a variant or engineered versionof a naturally occurring polypeptide. Parent polypeptide may refer tothe polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant an unmodified Fcpolypeptide that is modified to generate a variant, and by “parentantibody” as used herein is meant an unmodified antibody that ismodified to generate a variant antibody.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or V_(H) genes that make up the κ, λ,and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide” as used herein is meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification. Variant polypeptide may refer to thepolypeptide itself, a composition comprising the polypeptide, or theamino sequence that encodes it. Preferably, the variant polypeptide hasat least one amino acid modification compared to the parent polypeptide,e.g. from about one to about ten amino acid modifications, andpreferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent polypeptidesequence, and most preferably at least about 90% homology, morepreferably at least about 95% homology. Accordingly, by “Fc protein” asused herein is meant an Fc sequence that differs from that of a parentFc sequence by virtue of at least one amino acid modification. An Fcprotein may only encompass an Fc region, or may exist in the context ofan antibody, Fc fusion, or other polypeptide that is substantiallyencoded by Fc. Fc protein may refer to the Fc polypeptide itself,compositions comprising the Fc protein polypeptide, or the amino acidsequence that encodes it. In an exemplary embodiment, the variantproteins described herein comprise an Fc protein, as described herein,and as such, may comprise an antibody (and the correspondingderivatives) with the Fc protein, or an Fc fusion protein that comprisesthe Fc protein. In addition, in some cases, the Fc is a variant ascompared to a wild-type Fc, or to a “parent” variant.

The term “Antibody-Dependent Cell-Mediated Cytotoxicity” (ADCC) refersto a mechanism of cell-mediated immunity whereby an effector cell of theimmune system actively lyses a target cell that has been bound byspecific antibodies. The Fc region Variant polypeptides and proteinsdescribed herein successfully modulate ADCC as compared to the wild typeFc region antibodies. In certain embodiments, the Variant polypeptideresults in reduced ADCC as compared to the wild-type Fc region. Incertain other embodiments, the Fc region Variant described hereinresults in increased ADCC as compared to the corresponding wild-type Fcregion.

The term geometric metrics describes the plurality of measurablephysical attributes of a structural unit or residue (usedinterchangeably herein) of a biopolymer, an amino acid in the case of aprotein. Geometric metrics can be defined either by 1) dihedral, planeor other measurable angles as defined by the atoms of the respectiveresidue, 2) distances between atoms of the same residue or to atoms ofanother residue, and/or 3) distance between atoms of the same residueand a reference atom or position in the structure.

A residue population is the sum of all snapshots or samples obtained fora single residue. In certain embodiments the population is equal to thenumber of frames captured in a molecular dynamics simulation or thenumber of samples taken from a Monte Carlo simulation.

A residue conformation is defined in terms of the observed combinationof geometric metrics attributed to a particular residue structure. Aresidue cluster refers to the plurality of snapshots, which have beenassigned the same conformation, based on clustering all or a part of thedefined geometric metrics. A conformational frequency refers to thenumber of frames which have been assigned the same residue conformation.

Simulation refers to the process of conformational sampling, performedeither by either molecular dynamics or a Monte Carlo based samplingapproach. A trajectory contains the conformational frames produced by asimulation. Graph theory refers to the term as used in mathematics andcomputer science (for example, see Reinhard Diestel, Graph Theory;Edition 3, Springer 2005).

The term clustering tools refers to the plurality of mathematicalmethods and algorithms and programs which implement those, that can beused to identify clusters of similar data points from data sets.

The term Dynamical Cross Correlation Method refers to a graphicalrepresentation of the cross correlation matrix of atomic displacementsof a molecular structure in a molecular dynamics trajectory withreference to another conformational state of that structure.

Normal mode analysis is the study of characteristic harmonic vibrationsand frequencies about a local energy minimum of a molecular system.

Elastic network model refers to the representation of a proteinstructure as comprising of a network of harmonic springs approximatingthe interaction between residue pairs.

Provided herein are polypeptides comprising a variant Fc region, whereinsaid variant Fc region comprises at least three amino acid modificationsrelative to a wild-type Fc region, and has an altered effect relative toa polypeptide comprising a wild-type Fc region or variant Fc regioncomprising only one or two amino acid modifications; and wherein atleast two of the modifications provide a synergistic effect compared tosingle position modifications thereby exhibiting a selected bindingprofile to Fcγ receptors. In certain embodiments, one or both amino acidmodifications are located between positions 234-330 according to the EUindex. In certain embodiments, the modifications do not comprisesimultaneous substitution at positions 317 and 353 according to the EUindex. In some embodiments, the amino acid modifications do not comprisea substitution at position 332 according to the EU index. In certainembodiments, the polypeptides comprise the modificationsL235A/S239E/D265E. In some embodiments, the polypeptides comprise themodifications A327H/E269L/K236A. In one embodiment, the polypeptidescomprise the modifications G237F/D270Q/S239E. In some other embodiments,the polypeptides comprise the modifications A330V/I332L/K326. In oneembodiment, the polypeptides comprise the modificationsG236S/A327H/A330I.

In certain embodiments of the polypeptides described herein, the aminoacid modifications produce amino acid interactions and dynamics thatresult in enhanced binding affinity and/or specificity to a first Fcγreceptor while diminishing binding affinity and/or specificity to asecond Fcγ receptor compared to a polypeptide that lacks the three ormore amino acid modifications. In some of these embodiments, the firstFcγ receptor is FcγRIIIa receptor and the second Fcγ receptor is FcγRIIaor FcγRIIb. In certain embodiments, the amino acid modifications producefavorable FcγRIIIa-specific interactions and/or unfavorable interactionswith FcγRIIa and/or FcγRIIb receptors. In some embodiments, the aminoacid modifications have minimal impact on the FcγRIIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIaand/or FcγRIIb.

Provided herein are polypeptides comprising a variant Fc region, whereinsaid variant Fc region comprises at least three amino acid modificationsrelative to a wild-type Fc region, wherein the amino acid modificationsproduce amino acid interactions and dynamics that result in enhancedbinding affinity and/or specificity to a FcγRIIIa receptor whilediminishing binding affinity and/or specificity to FcγRIIa or FcγRIIbreceptor compared to a polypeptide that lacks the two amino acidmodifications. In some embodiments, the polypeptide comprisesmodifications S239E/D265S/1332E. In certain embodiments, the polypeptidecomprises the modifications G237F/S239E/A327H. In certain otherembodiments, the polypeptide comprises modificationsH268D/E269L/S298A/K326A/A327H. In some embodiments, the polypeptidecomprises the modifications L235A/S239E/D265E/A327H. In an embodiment,the polypeptide comprises the modifications G237F/S239E/D270N. In someembodiments, the polypeptide comprises the modificationsG236E/G237F/S239E. In an embodiment, the polypeptide comprises themodifications S239E/D265SI332E and alternatively H268D. In certainembodiments, the polypeptide comprises modifications selected from thegroup of G237F/S239E/D265E, S239E/S298A/K326A/A327H, andG236E/D270N/A327V/I332E. In certain embodiments, the polypeptidecomprises the modifications S298A/K326A/A327H wherein the polypeptidehas improved binding selectivity to FcγRIIIa receptor as compared to apolypeptide lacking the S298A/K326A/A327H modifications.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fe region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein the aminoacid modifications produce amino acid interactions and dynamics thatresult in enhanced binding affinity and/or specificity to a FcγRIIareceptor while diminishing binding affinity and/or specificity toFcγRIIIa or FcγRIIb receptor compared to a polypeptide that lacks atleast one of the amino acid modifications. In some embodiments, thepolypeptide comprises modifications G237F, A327L and A330I. In certainembodiments, the amino acid modifications produce favorableFcγRIIa-specific interactions and/or unfavorable interactions withFcγRIIIa and/or FcγRIIb receptors. In certain embodiments, the aminoacid modifications have minimal impact on the FcγRIIa receptor whileproducing detrimental effects on binding of the polypeptide to FcγRIIIaand/or FcγRIIb. In certain embodiments, the polypeptide comprisesmodifications G237F, S239E and H268D. In some embodiments, thepolypeptide comprises modifications D265E/S267D/A330S.

In a further aspect, provided herein are polypeptides comprising avariant Fc region, wherein said variant Fc region comprises at leastthree amino acid modifications relative to a wild-type Fc region,wherein the amino acid modifications produce amino acid interactions anddynamics that result in enhanced binding affinity and/or specificity toa FcγRIIb receptor while diminishing binding affinity and/or specificityto FcγRIIIa or FcγRIIa receptor compared to a polypeptide that lacks atleast one of the amino acid modifications. In certain embodiments, theamino acid modifications produce favorable FcγRIIb-specific interactionsand/or unfavorable interactions with FcγRIIIa and/or FcγRIIa receptors.In certain embodiments, the amino acid modifications have minimal impacton the FcγRIIb receptor while producing detrimental effects on bindingof the polypeptide to FcγRIIIa and/or FcγRIIa. In certain embodiments,the polypeptide comprises modifications S239D/D265S/S298A/I332E. In someother embodiments, the polypeptide comprises the modificationsG237F/S298A/A330L/I332E. In some embodiments the polypeptide comprisesthe modifications H268D, K326A, A327H and alternatively one or both ofE269L and S298A. In an embodiment, the polypeptide comprises themodifications G237F/V266L/S267D. In some embodiments, the polypeptidescomprise the modifications L234F/S267G/N325L or L234F/S267E/N325L. In anembodiment, the polypeptide comprises modificationsG236A/S239D/D270L/I332E.

In certain aspects described herein, the binding of the polypeptidecomprising a wild-type Fc region to Fcγ receptors is detectable by an invitro assay.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein one of themodifications comprises the mutation S239E wherein the polypeptide hashigher selectivity in binding to the FcγRIIIa receptor compared to apolypeptide that lacks the S239E mutation.

In an aspect, provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein one of themodifications comprises the mutation S239E one of the modificationscomprises the mutation S298A wherein the polypeptide has reduced bindingaffinity to FcγRIIa and FcγRIIb receptors compared to a polypeptide thatlacks the S298A mutation.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, said modificationsselected from D270L/Y300L/A330K, G237F/S267G/N325F, G237F/V266L/S267D,L234F/S267G/N325L, L234F/S267E/N325L, G237F/S239E/A327H,G237F/A327L/A330I, S239E/A327L/A330I, S239E/S267E/H268D,G237F/S239E/D270N, G236E/G237F/S239E, S239E/D265S/I332E,G237F/S239E/D265E, G237F/S239E/H268D, H268E/D270E/S267G,H268D/K326A/A327H, D265E/S267D/A330S, L235A/S239E/D265E,A327H/E269L/K236A, G237F/D270Q/S239E, A330V/I332L/K326, andG236S/A327H/A330I.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least four aminoacid modifications relative to a wild-type Fc region, said modificationsselected from L235A/S239E/D265E/A327H, S239E/D265S/H268D/I332E,S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H,G237F/S298A/A330L/I332E, and G236E/D270N/A327V/I332E,G236A/S239D/D270L/I332E and H268D/E269L/S298A/K326A/A327H.

In an aspect provided herein are polypeptides comprising a variant Fcregion, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region, wherein the atleast three amino acid modifications are selected from the groupconsisting of: L234Q, L234N, L235A, G236E, E236L, E236D, G237F, G237N,S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268D, H268E, E269L,E269L, D270N, D270I, D270E, S298A, K326A, K326D, A327H, A327V, A327L,A327T, A330V, A330L, A330W, A330I, A330S, I332L, I332D, and 1332E.

In an aspect provided herein are polypeptides comprising a variant Fcregion, said variant Fc region comprises a combination of amino acidmodifications wherein said combination is selected from the groupconsisting of: L235A/S239E/D265E; L235A/G237F/D265E; S239E/E269D/A327H;S239E/G237N/A327H; S239E/G237F/A327V; G237F/D270I/S239E;G237F/A327L/S239E; A327H/E269L/K326A; A330V/I332L/S239E;A327T/E269L/K326A; D270N/A327T/K326A; A330V/I332L/S239E;A330W/I332D/S239E; G236E/D265E/A327H/A330I; D270N/S298A/A327V;G236E/D265E/D270N/A327H/A330I; G236E/D270N/A327H/A330I;G236E/D270N/A327V/I332E; G236E/D270N/A327V/G237F;L234N/S239E/A330I/I332E; L234Q/S239E/A330I/I332E;L234Q/S239E/A330I/I332E/S298A; G237F/S239D/D265E/D270N/S298A;G237F/S239E/D270N/A330L/I332E; G237F/S239E/D270N/A330L/I332E/S298A;S239E/G237F/A327H; G237F/A327L/A330I; S239E/A330I/A327L;D265E/S239E/L235A/A327H; S267E/S239E/H268D; G237F/D270N/S239E;S239E/G237F/G236E; I332E/D265S/S239E/H268D; I332E/D265S/S239E;D265E/S239E/G237F; S239E/H268D/G237F; S298A/D265S/S239D/I332E;S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E; H268E/D270E/S267G;H268D/K326A/A327H; H268D/K326A/A327H/E269L/S298A; A330S/D265E/S267D;S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G

In an aspect, provided herein are polypeptides that comprise a variantFc region, wherein said variant Fc region comprises at least three aminoacid modifications relative to a wild-type Fc region wherein when saidvariant Fc region comprises amino acid modification H268D, said variantdoes not comprise the modification S267E.

In certain embodiments of the polypeptides described herein, the Fcregion of the parent polypeptide is a human IgG Fc region. In some ofthese embodiments, the human IgG Fc region is a human IgG1, IgG2, IgG3,or IgG4 Fc region.

In certain embodiments of the polypeptides described herein, thepolypeptide is an antibody. In some embodiments, the antibody is amonoclonal antibody, a humanized antibody, or a human antibody.

In an aspect is a nucleic acid comprising: a nucleotide sequenceencoding a polypeptide described herein. In certain embodiments is avector, comprising the nucleic acid.

In an aspect is a method for producing a polypeptide or proteindescribed herein, said method comprising: (i) culturing in a medium ahost cell comprising a nucleic acid encoding said polypeptide, underconditions suitable for the expression of said polypeptide; and (ii)recovering the polypeptide from said medium.

In an aspect described herein is a therapeutic antibody thatspecifically binds a cancer target antigen, said therapeutic antibodycomprising a variant Fc region polypeptide described herein. In certainembodiments, the therapeutic antibody is selected from the groupconsisting of abagovomab, adalimumab, alemtuzumab, aurograb,bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab,canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab,denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab,ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab,motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab,ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab,ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab,tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab,zalutumumab, and any other antibodies. In certain embodiments, thetarget antigen is selected from the group consisting of a-chain (CD25)of IL-2R, Amyloid beta, anti-EpCAM×anti-CD3, BLyS (or BAFF), CD11a,CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F proteinof RSV, G250, glycoprotein IIb/IIIa R, HER2, HER2/neu R, Hsp90, IgEantibody, IL-12/IL-23, IL-1b, IL-5, IL-6 receptor, Integrinalpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF alpha, VEGF-A, and othertherapeutically advantageous targets.

In an aspect described herein is a method of treating cancer in apatient having a cancer characterized by a cancer antigen, said methodcomprising administering to said patient a therapeutically effectiveamount of a therapeutic antibody described herein. In certainembodiments, the patient is human.

In an aspect described herein is a method of treating immune disordersin a patient having an immune disorder characterized by an immuneantigen, said method comprising administering to said patient atherapeutically effective polypeptide, antibody or protein describedherein.

In an aspect is a pharmaceutical composition, said compositioncomprising a therapeutically effective amount of a polypeptide describedherein, and a pharmaceutically acceptable carrier.

In an aspect described herein are polypeptides, said polypeptidescomprising a variant Fc region with at least three amino acidsubstitutions, and wherein said polypeptides are more effective atmediating antibody-dependent cellular cytotoxicity (ADCC) relative towild type. In certain embodiments, the polypeptide comprising a variantFc region is about 1.5 to about 100 fold more effective in mediatingADCC relative to wild type. In certain embodiments, the polypeptidecomprising a variant Fc region is about 2 to about 50 fold moreeffective in mediating ADCC relative to wild type.

In an aspect described herein are polypeptides, said polypeptidescomprising a variant Fc region with at least three amino acidsubstitutions, wherein the polypeptide is more effective at mediatinginhibition of inflammatory immune responses relative to wild type. Incertain embodiments, the polypeptide comprising a variant Fc region isabout 2 fold more effective in mediating inhibition of inflammatoryimmune responses relative to wild type. In some embodiments, thepolypeptide comprising a variant Fc region is about 10 fold moreeffective in mediating inhibition of inflammatory immune responsesrelative to wild type. In certain embodiments, the polypeptidecomprising a variant Fc region is about 50 fold more effective inmediating inhibition of inflammatory immune responses relative to wildtype. In certain embodiments, the polypeptide comprising a variant Fcregion is about 100 fold more effective in mediating inhibition ofinflammatory immune responses relative to wild type.

Also provided herein is a method for identifying Fc variant polypeptidesin silico based on calculated binding affinities to FcγRIIa, FcγRIIband/or FcγRIIIa. In certain embodiments, the method of identifying Fcvariant polypeptides in silico further calculates in silicoelectrostatics, solvation, packing, packing density, hydrogen binding,and entropic effects of said Fc variant polypeptides. In certainembodiments, the method of identifying Fc variant polypeptides in silicofurther comprises constructing the identified Fc variant polypeptidesand expressing said polypeptides in the context of an antibody inmammalian cells.

The Fc polypeptides and proteins described herein may be optimized for avariety of properties. Properties that may be optimized include but arenot limited to enhanced or reduced affinity for an FcγR. In a preferredembodiment, the Fc proteins described herein are optimized to possessenhanced affinity for a human activating FcγRI, preferably FcγRII,FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, most preferably FcγRIIIa. Inan alternately preferred embodiment, the Fc proteins are optimized topossess reduced affinity for the human inhibitory receptor FγRIIb. Thesepreferred embodiments are anticipated to provide antibodies and Fcfusions with enhanced therapeutic properties in humans, for exampleenhanced effector function and greater anti-cancer potency. In analternate embodiment, the Fc proteins described herein are optimized tohave reduced or ablated affinity for a human FcγRI, including but notlimited to FcγRII, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.These embodiments are anticipated to provide antibodies and Fc fusionswith enhanced therapeutic properties in humans, for example reducedeffector function and reduced toxicity. Preferred embodiments compriseoptimization of Fc binding to a human FcγR, however in alternateembodiments the Fc polypeptides and proteins of the present inventionpossess enhanced or reduced affinity for FcγRs from nonhuman organisms,including but not limited to mice, rats, rabbits, and monkeys. Fcproteins that are optimized for binding to a nonhuman FcγR may find usein experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies or Fc fusions that comprise Fc proteins that are optimizedfor one or more mouse FcγRs, may provide valuable information withregard to the efficacy of the antibody or Fc fusion, its mechanism ofaction, and the like. In certain embodiments, the Fc polypeptides andproteins described herein are optimized for enhanced functionalityand/or solution properties in aglycosylated form. In an exemplaryembodiment, the aglycosylated Fc proteins described herein bind an Fcligand with greater affinity than the aglycosylated form of the parentFc polypeptide. Said Fc ligands include but are not limited to FcγRs,C1q, FcRn, and proteins A and G, and may be from any source includingbut not limited to human, mouse, rat, rabbit, or monkey, preferablyhuman. In an alternately preferred embodiment, the Fc proteins areoptimized to be more stable and/or more soluble than the aglycosylatedform of the parent Fc polypeptide. An Fc protein that is engineered orpredicted to display any of the aforementioned optimized properties isherein referred to as an “optimized Fc protein”.

In certain embodiments, the Fc polypeptides and proteins describedherein are derived from parent Fc polypeptides that are themselves froma wide range of sources. In some embodiments the parent Fc polypeptideis substantially encoded by one or more Fc genes from any organism,including but not limited to humans, mice, rats, rabbits, camels,llamas, dromedaries, monkeys, preferably mammals and most preferablyhumans and mice. In an embodiment, the parent Fc polypeptide comprisesan antibody, referred to as the parent antibody. In certain embodiments,the parent antibody is fully human, obtained for example usingtransgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol8:455-458) or human antibody libraries coupled with selection methods(Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108). The parentantibody need not be naturally occurring. In certain embodiments theparent antibody is an engineered antibody, including but not limited tochimeric antibodies and humanized antibodies (Clark, 2000, Immunol Today21:397-402). In certain embodiments, the parent antibody is anengineered variant of an antibody that is substantially encoded by oneor more natural antibody genes. In one embodiment, the parent antibodyhas been affinity matured, as is known in the art. Alternatively, theantibody has been modified in some other way, for example as describedin U.S. Ser. No. 10/339,788, filed on Mar. 3, 2003.

In certain embodiments, the Fc proteins described herein issubstantially encoded by immunoglobulin genes belonging to any of theantibody classes. In an exemplary embodiment, the Fc proteins describedherein find use in antibodies or Fc fusions that comprise sequencesbelonging to the IgG class of antibodies, including IgG1, IgG2, IgG3, orIgG4. In an alternate embodiment the Fc proteins described herein finduse in antibodies or Fc fusions that comprise sequences belonging to theIgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classesof antibodies. In some embodiments, the Fc proteins described hereincomprise more than one protein chain. That is, in some embodiments, thepolypeptides described herein find use in an antibody or Fc fusion thatis a monomer or an oligomer, including a homo- or hetero-oligomer.

In some embodiments, the antibodies of the invention are based on humansequences, and thus human sequences are used as the “base” sequences,against which other sequences, such as rat, mouse, and monkey sequencesare compared. In order to establish homology to primary sequence orstructure, the amino acid sequence of a precursor or parent Fcpolypeptide is directly compared to the human Fc sequence outlinedherein. After aligning the sequences, using one or more of the homologyalignment programs known in the art (for example using conservedresidues as between species), allowing for necessary insertions anddeletions in order to maintain alignment (i.e., avoiding the eliminationof conserved residues through arbitrary deletion and insertion), theresidues equivalent to particular amino acids in the primary sequence ofhuman Fc are defined. Alignment of conserved residues preferably shouldconserve 100% of such residues. However, alignment of greater than 75%or as little as 50% of conserved residues is also adequate to defineequivalent residues (sometimes referred to as “corresponding residues”).Equivalent residues may also be defined by determining homology at thelevel of tertiary structure for an Fc polypeptide whose tertiarystructure has been determined. Equivalent residues are defined as thosefor which the atomic coordinates of two or more of the main chain atomsof a particular amino acid residue of the parent or precursor (N on N,CA on CA, C on C and 0 on 0) are within 0.13 nm and preferably 0.1 nmafter alignment. Alignment is achieved after the best model has beenoriented and positioned to give the maximum overlap of atomiccoordinates of non-hydrogen protein atoms of the Fc polypeptide.

In some embodiments, the Fc polypeptides described herein are combinedwith other Fc modifications, including but not limited to modificationsthat alter effector function or interaction with one or more Fc ligands.In certain embodiments, these combinations provide additive,synergistic, or properties in antibodies or Fc fusions. In oneembodiment, the Fc proteins described herein is combined with otherknown Fc proteins (Duncan et al., 1988, Nature 332:563-564; Lund et al.,1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59;Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al.,1995, Proc Natl Acad Sci USA 92:11980-11984; Jefferis et al., 1995,Immunol Left 44:111-117; Lund et al., 1995, Faseb J9:115-119; Jefferiset al., 1996, Immunol Left 54:101-104; Lund et al., 1996, J Immunol157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogieet al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al.,2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65; Presta etal., 2002, Biochem Soc Trans 30:487-490; Hinton et al., 2004, J BiolChem 279:6213-6216) (U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; US2004/0002587 A1). In an alternate embodiment, the Fc proteins describedherein are incorporated into an antibody or Fc fusion that comprises oneor more engineered glycoforms. By “engineered glycoform” as used hereinis meant a carbohydrate composition that is covalently attached to an Fcpolypeptide, wherein said carbohydrate composition differs chemicallyfrom that of a parent Fc polypeptide. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. Engineered glycoforms may be generated bya variety of methods known in the art (Umana et al., 1999, NatBiotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S.Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1); (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [GLYCART biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an Fc polypeptidein various organisms or cell lines, engineered or otherwise (for exampleLec-13 CHO cells or rat hybridoma YB2/0 cells), by regulating enzymesinvolved in the glycosylation pathway (for example FUT8[.alpha.1,6-fucosyltranserase] and/or.beta.1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the Fc polypeptide has been expressed. Engineeredglycoform typically refers to the different carbohydrate oroligosaccharide; thus an Fc polypeptide, for example an antibody or Fcfusion, may comprise an engineered glycoform. Alternatively, engineeredglycoform may refer to the Fc polypeptide that comprises the differentcarbohydrate or oligosaccharide. Thus combinations of the Fc proteinsdescribed herein with other Fc modifications, as well as undiscovered Fcmodifications, are contemplated with the goal of generating antibodiesor Fc fusions with optimized properties.

In certain embodiments, the variant Fc proteins and polypeptidesdescribed herein find use in an antibody. By “antibody described herein”as used herein is meant an antibody that comprises an Fc proteindescribed herein. The present invention may, in fact, find use in anyprotein that comprises Fc, and thus application of the Fc polypeptideand proteins described herein is not limited to antibodies. The Fcproteins described herein may find use in an Fc fusion. By “Fc fusiondescribed herein” as used herein refers to an Fc fusion that comprisesan Fc protein described herein. Fc fusions may comprise an Fc proteindescribed herein operably linked to a cytokine, soluble receptor domain,adhesion molecule, ligand, enzyme, peptide, or other protein or proteindomain, and include but are not limited to Fc fusions described in U.S.Pat. No. 5,843,725; U.S. Pat. No. 6,018,026; U.S. Pat. No. 6,291,212;U.S. Pat. No. 6,291,646; U.S. Pat. No. 6,300,099; U.S. Pat. No.6,323,323; PCT WO 00/24782; and in (Chamow et al., 1996, TrendsBiotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200).

Virtually any antigen may be targeted by the proteins and polypeptidesdescribed herein, including but not limited to the following list ofproteins, subunits, domains, motifs, and epitopes belonging to thefollowing list of proteins: CD2; CD3, CD3E, CD4, CD11, CD11a, CD14,CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33(p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3,IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15,IL-18, IL-23, interferon alpha, interferon beta, interferon gamma;TNF-alpha, TNFbeta2, TNFc, TNFalphabeta, TNF-RI, TNF-RII, FasL, CD27L,CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L,TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin Beta Receptor,TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, integrin beta1,integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin alpha3,integrin alpha4, integrin alpha5, integrin alpha6, integrin alphav,alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4,L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor,B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS(B-lymphocyte Stimulator), complement C5, IgE, factor VII, CD64, CBL,NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4),Tissue Factor, VEGF, VEGFR, endothelin receptor, VLA-4, Hapten NP-cap orNIP-cap, T cell receptor alpha/beta, E-selectin, digoxin, placentalalkaline phosphatase (PLAP) and testicular PLAP-like alkalinephosphatase, transferrin receptor, Carcinoembryonic antigen (CEA),CEACAM5, HMFG PEM, mucin MUC1, MUC18, Heparanase 1, human cardiacmyosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associatedantigen CA 125, Prostate specific membrane antigen (PSMA), Highmolecular weight melanoma-associated antigen (HMW-MAA),carcinoma-associated antigen, Gcoprotein IIb/IIIa (GPIIb/IIIa),tumor-associated antigen expressing Lewis Y related carbohydrate, humancytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV,respiratory syncital virus RSV F, RSVF Fgp, VNRintegrin, IL-8,cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIVHIB gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes simplexvirus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelopeglycoprotein, and Clostridium perfringens toxin.

One skilled in the art will appreciate that the aforementioned list oftargets refers not only to specific proteins and biomolecules, but thebiochemical pathway or pathways that comprise them. For example,reference to CTLA-4 as a target antigen implies that the ligands andreceptors that make up the T cell co-stimulatory pathway, includingCTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands orreceptors that bind these proteins, are also targets. Thus target asused herein refers not only to a specific biomolecule, but the set ofproteins that interact with said target and the members of thebiochemical pathway to which said target belongs. One skilled in the artwill further appreciate that any of the aforementioned target antigens,the ligands or receptors that bind them, or other members of theircorresponding biochemical pathway, may be operably linked to the Fcproteins described herein in order to generate an Fc fusion. Thus forexample, an Fc fusion that targets EGFR could be constructed by operablylinking an Fc protein to EGF, TGF.alpha., or any other ligand,discovered or undiscovered, that binds EGFR. Accordingly, an Fc proteindescribed herein could be operably linked to EGFR in order to generatean Fc fusion that binds EGF, TGF.alpha., or any other ligand, discoveredor undiscovered, that binds EGFR. Thus virtually any polypeptide,whether a ligand, receptor, or some other protein or protein domain,including but not limited to the aforementioned targets and the proteinsthat compose their corresponding biochemical pathways, may be operablylinked to the Fc proteins described herein to develop an Fc fusion.

A number of antibodies and Fc fusions that are approved for use, inclinical trials, or in development benefit from the Fc proteinsdescribed herein. Said antibodies and Fc fusions are herein referred toas “clinical products and candidates”. Thus in a preferred embodiment,the Fc proteins described herein may find use in a range of clinicalproducts and candidates. For example, a number of antibodies that targetCD20 may benefit from the Fc proteins described herein. For example theFc proteins described herein may find use in an antibody that issubstantially similar to rituximab (Rituxan®, IDEC/Genentech/Roche) (seefor example U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibodyapproved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20currently being developed by Genmab, an anti-CD20 antibody described inU.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20(Immunomedics, Inc.), and HumaLYM (Intracel). A number of antibodiesthat target members of the family of epidermal growth factor receptors,including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4(ErbB-4), may benefit from the Fc proteins described herein. For examplethe Fc proteins described herein may find use in an antibody that issubstantially similar to trastuzumab (Herceptin®, Genentech) (see forexample U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibodyapproved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®),currently being developed by Genentech; an anti-Her2 antibody describedin U.S. Pat. No. 4,753,894; cetuximab (Erbitux®), Imclone) (U.S. Pat.No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody inclinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No.6,235,883), currently being developed by Abgenix/Immunex/Amgen;HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed byGenmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat.No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60;Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al.,1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research)(PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993,22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993,67(2):247-53; Modjtahedi et al., 1996, Br J Cancer, 73(2):228-35;Modjtahedi et al., 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YMBiosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat.No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al., 1997,Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institue for CancerResearch, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc NatlAcad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX,National Cancer Institute) (PCT WO 0162931A2); and SC 100 (Scancell)(PCT WO 01/88138). In another preferred embodiment, the Fc proteinsdescribed herein may find use in alemtuzumab (Campath®, Millenium), ahumanized monoclonal antibody currently approved for treatment of B-cellchronic lymphocytic leukemia. The Fc proteins described herein may finduse in a variety of antibodies or Fc fusions that are substantiallysimilar to other clinical products and candidates, including but notlimited to muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibodydeveloped by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan(Zevalin®), an anti-CD20 antibody developed by IDEC/Schering AG,gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibodydeveloped by Celltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fcfusion developed by Biogen), abciximab (ReoPro®), developed byCentocor/Lilly, basiliximab (Simulect®), developed by Novartis,palivizumab (Synagis®), developed by Medlmmune, infliximab (Remicade®),an anti-TNFalpha antibody developed by Centocor, adalimumab (Humira®),an anti-TNFalpha antibody developed by Abbott, Humicade®, ananti-TNFalpha antibody developed by Celltech, etanercept (Enbrel®), ananti-TNFalpha Fc fusion developed by Immunex/Amgen, ABX-CBL, ananti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8antibody being developed by Abgenix, ABX-MA1, an anti-MUC18 antibodybeing developed by Abgenix, Pemtumomab (R1549, .sup.90Y-muHMFG1), ananti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1antibody being developed by Antisoma, AngioMab (AS1405), being developedby Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407)being developed by Antisoma, Antegren®) (natalizumab), ananti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developedby Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed byBiogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibodybeing developed by Biogen, CAT-152, an anti-TGF.beta.2 antibody beingdeveloped by Cambridge Antibody Technology, J695, an anti-IL-12 antibodybeing developed by Cambridge Antibody Technology and Abbott, CAT-192, ananti-TGF.beta.1 antibody being developed by Cambridge AntibodyTechnology and Genzyme, CAT-213, an anti-Eotaxin1 antibody beingdeveloped by Cambridge Antibody Technology, LymphoStat-B® an anti-Blysantibody being developed by Cambridge Antibody Technology and HumanGenome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1 antibody beingdeveloped by Cambridge Antibody Technology and Human Genome Sciences,Inc., Avastin® (bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody beingdeveloped by Genentech, an anti-HER receptor family antibody beingdeveloped by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factorantibody being developed by Genentech, Xolair® (Omalizumab), an anti-IgEantibody being developed by Genentech, Raptiva® (Efalizumab), ananti-CD1a antibody being developed by Genentech and Xoma, MLN-02Antibody (formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase 1 antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide® (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem® (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNF.alpha. antibodybeing developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF®, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-.quadrature.5.quadrature.1 Integrin, beingdeveloped by Protein Design Labs, anti-IL-12, being developed by ProteinDesign Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma.

Application of the Fc polypeptides, proteins to the aforementionedantibody and Fc fusion clinical products and candidates is not meant tobe constrained to their precise composition. In some embodiments, the Fcproteins described herein are incorporated into the aforementionedclinical candidates and products, or into antibodies and Fc fusions thatare substantially similar to them. In certain embodiments, the Fcproteins described herein are incorporated into versions of theaforementioned clinical candidates and products that are humanized,affinity matured, engineered, or modified in some other way.Furthermore, the entire polypeptide of the aforementioned clinicalproducts and candidates need not be used to construct a new antibody orFc fusion that incorporates the Fc proteins described herein; forexample only the variable region of a clinical product or candidateantibody, a substantially similar variable region, or a humanized,affinity matured, engineered, or modified version of the variable regionmay be used. In another embodiment, the Fc proteins and polypeptidesdescribed herein are used in an antibody or Fc fusion that binds to thesame epitope, antigen, ligand, or receptor as one of the aforementionedclinical products and candidates.

In an aspect the Fc polypeptides described herein are used in a widerange of antibody and Fc fusion products. In one embodiment the antibodyor Fc fusion comprising the Fc polypeptide or protein described hereinis a therapeutic, a diagnostic, or a research reagent, preferably atherapeutic. In certain other embodiments, the antibodies and Fc fusionscomprise the Fc based polypeptides described herein and are used foragricultural or industrial uses. In an alternate embodiment, the Fcproteins and polypeptides described herein compose a library that isscreened experimentally. In certain embodiments, this library comprisesa list of nucleic acid or amino acid sequences. In certain otherembodiments, the library is a physical composition of nucleic acids orpolypeptides that encode the library sequences. In some embodiments, theFc proteins find use in an antibody composition that is monoclonal orpolyclonal. The antibodies and Fe fusions described herein encompass,but are not restricted to agonists, antagonists, neutralizing,inhibitory, or stimulatory. In an exemplary embodiment, the antibodiesand Fc fusions described herein are used to kill target cells that bearthe target antigen, for example cancer cells. In an alternateembodiment, the antibodies and Fc fusions described herein are used toblock, antagonize, or agonize the target antigen, for example forantagonizing a cytokine or cytokine receptor. In an alternateembodiment, the antibodies and Fc fusions described herein are used toblock, antagonize, or agonize the target antigen and kill the targetcells that bear the target antigen.

In some embodiments, the polypeptides disclosed herein are useful inregulating the immune response, e.g., in inhibiting the immune responsein connection with autoimmune diseases or inflammatory diseases. Suchpolypeptides have therapeutic utility in treating and/or preventing anautoimmune disorder. Examples of autoimmune disorders that may betreated by administering the polypeptides of the disclosed hereininclude, but are not limited to, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgAneuropathy, juvenile arthritis, lichen planus, lupus erthematosus,Meniere's disease, mixed connective tissue disease, multiple sclerosis,type 1 or immune-mediated diabetes mellitus, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld'sphenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupuserythematosus, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, and chronic inflammation resulting from chronicviral or bacteria infections. Examples of inflammatory disorders whichcan be prevented, treated or managed in accordance with the methods ofthe invention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

The Fc polypeptides described herein are used for various therapeuticpurposes. In some embodiments, the Fc polypeptides and proteins areadministered to a patient to treat an antibody-related disorder. A“patient” for the purposes described herein includes both humans andother animals, preferably mammals and most preferably humans. Thus theantibodies and Fc fusions described herein have both human therapy andveterinary applications. In the preferred embodiment the patient is amammal, and in the most preferred embodiment the patient is human. Theterm “treatment” in the present invention is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor a disease or disorder. Thus, for example, successful administrationof an antibody or Fc fusion prior to onset of the disease results intreatment of the disease. As another example, successful administrationof an optimized antibody or Fc fusion after clinical manifestation ofthe disease to combat the symptoms of the disease comprises treatment ofthe disease. “Treatment” also encompasses administration of an optimizedantibody or Fc fusion protein after the appearance of the disease inorder to eradicate the disease. Successful administration of an agentafter onset and after clinical symptoms have developed, with possibleabatement of clinical symptoms and perhaps amelioration of the disease,comprises treatment of the disease. Those “in need of treatment” includemammals already having the disease or disorder, as well as those proneto having the disease or disorder, including those in which the diseaseor disorder is to be prevented. By “antibody related disorder” or“antibody responsive disorder” or “condition” or “disease” herein aremeant a disorder that may be ameliorated by the administration of apharmaceutical composition comprising an antibody or Fc fusion describedherein. Antibody related disorders include but are not limited toautoimmune diseases, immunological diseases, infectious diseases,inflammatory diseases, neurological diseases, and oncological andneoplastic diseases including cancer. By “cancer” and “cancerous” hereinrefer to or describe the physiological condition in mammals that istypically characterized by unregulated cell growth. Examples of cancerinclude but are not limited to carcinoma, lymphoma, blastoma, sarcoma(including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma,meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophageal cancer, tumors of the biliary tract, as well as head and neckcancer. Furthermore, the Fc proteins described herein may be used totreat conditions including but not limited to congestive heart failure(CHF), vasculitis, rosacea, acne, eczema, myocarditis and otherconditions of the myocardium, systemic lupus erythematosus, diabetes,spondylopathies, synovial fibroblasts, and bone marrow stroma; boneloss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer;disuse osteopenia; malnutrition, periodontal disease, Gaucher's disease,Langerhans' cell histiocytosis, spinal cord injury, acute septicarthritis, osteomalacia, Cushing's syndrome, monoostotic fibrousdysplasia, polyostotic fibrous dysplasia, periodontal reconstruction,and bone fractures; sarcoidosis; multiple myeloma; osteolytic bonecancers, breast cancer, lung cancer, kidney cancer and rectal cancer;bone metastasis, bone pain management, and humoral malignanthypercalcemia, ankylosing spondylitisa and other spondyloarthropathies;transplantation rejection, viral infections, hematologic neoplasisas andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplamacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia,tumors of the central nervous system, e.g., brain tumors (glioma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, and tumors of the vascularsystem (angiosarcoma and hemangiopericytoma), osteoporosis, hepatitis,HIV, AIDS, spondylarthritis, rheumatoid arthritis, inflammatory boweldiseases (IBD), sepsis and septic shock, Crohn's Disease, psoriasis,schleraderma, graft versus host disease (GVHD), allogenic islet graftrejection, hematologic malignancies, such as multiple myeloma (MM),myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML),inflammation associated with tumors, peripheral nerve injury ordemyelinating diseases.

In one embodiment, an antibody or polypeptide described herein isadministered to a patient having a disease involving inappropriateexpression of a protein. Within the scope described herein this is meantto include diseases and disorders characterized by aberrant proteins,due for example to alterations in the amount of a protein present, thepresence of a mutant protein, or both. An overabundance may be due toany cause, including but not limited to overexpression at the molecularlevel, prolonged or accumulated appearance at the site of action, orincreased activity of a protein relative to normal. Included within thisdefinition are diseases and disorders characterized by a reduction of aprotein. This reduction may be due to any cause, including but notlimited to reduced expression at the molecular level, shortened orreduced appearance at the site of action, mutant forms of a protein, ordecreased activity of a protein relative to normal. Such anoverabundance or reduction of a protein can be measured relative tonormal expression, appearance, or activity of a protein, and saidmeasurement may play an important role in the development and/orclinical testing of the antibodies and Fc fusions described herein.

In one embodiment, an antibody or polypeptide described herein is theonly therapeutically active agent administered to a patient.Alternatively, the antibody or Fc fusion described herein isadministered in combination with one or more other therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, or othertherapeutic agents. Such molecules are suitably present in combinationin amounts that are effective for the purpose intended. The skilledmedical practitioner can determine empirically the appropriate dose ordoses of other therapeutic agents useful herein. The antibodies andpolypeptides described herein may be administered concomitantly with oneor more other therapeutic regimens. For example, an antibody orpolypeptide described herein may be administered to the patient alongwith chemotherapy, radiation therapy, or both chemotherapy and radiationtherapy. In one embodiment, the antibody or Fc fusion described hereinmay be administered in conjunction with one or more antibodies orpolypeptides, which may or may not comprise an Fc protein describedherein.

In one embodiment, the antibodies and polypeptides described herein areadministered with a chemotherapeutic agent. By “chemotherapeutic agent”as used herein is meant a chemical compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include but are not limitedto alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; thymidylate synthase inhibitor (such as Tomudex); cox-2inhibitors, such as celicoxib (CELEBREX®) or MK-0966 (VIOXX®); andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such as antiestrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY 117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

In certain embodiments, a chemotherapeutic or other cytotoxic agent isadministered as a prodrug. By “prodrug” as used herein is meant aprecursor or derivative form of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. See, for example Wilman, 1986, Biochemical SocietyTransactions, 615th Meeting Belfast, 14:375-382; and Stella et al.,“Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed DrugDelivery, Borchardt et al., (ed.): 247-267, Humana Press, 1985. Theprodrugs that may find use with the present invention include but arenot limited to phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use with the antibodies and Fc fusions describedherein include but are not limited to any of the aforementionedchemotherapeutic agents.

In some embodiments, the proteins and polypeptides described herein arecombined with other therapeutic regimens. For example, in oneembodiment, the patient to be treated with the antibody or Fc fusionalso receives radiation therapy. Radiation therapy can be administeredaccording to protocols commonly employed in the art and known to theskilled artisan. Such therapy includes but is not limited to cesium,iridium, iodine, or cobalt radiation. In some embodiments, the radiationtherapy is whole body irradiation, or directed locally to a specificsite or tissue in or on the body, such as the lung, bladder, orprostate. Typically, radiation therapy is administered in pulses over aperiod of time from about 1 to 2 weeks. In some embodiments, theradiation therapy is administered over longer periods of time. In oneembodiment, radiation therapy is administered to patients having headand neck cancer for about 6 to about 7 weeks. Optionally, the radiationtherapy is administered as a single dose or as multiple, sequentialdoses. The skilled medical practitioner can determine empirically theappropriate dose or doses of radiation therapy useful herein. Inaccordance with another embodiment of the invention, the antibody or Fcfusion described herein and one or more other anti-cancer therapies areemployed to treat cancer cells ex vivo. It is contemplated that such exvivo treatment may be useful in bone marrow transplantation andparticularly, autologous bone marrow transplantation. In certainembodiments, treatment of cells or tissue(s) containing cancer cellswith antibody or Fc fusion and one or more other anti-cancer therapies,such as described above, is employed to deplete or substantially depletethe cancer cells prior to transplantation in a recipient patient. IIcertain embodiments, the proteins and Fc fusions of the invention areemployed in combination with still other therapeutic techniques such assurgery.

In an alternate embodiment, the antibodies and polypeptides describedherein are administered with a cytokine. By “cytokine” as used herein ismeant a generic term for proteins released by one cell population thatact on another cell as intercellular mediators. Examples of suchcytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture, and biologically active equivalents of the native sequencecytokines.

A variety of other therapeutic agents find use for administration withthe Fc variant proteins and polypeptides described herein. In oneembodiment, the protein is administered with an anti-angiogenic agent.By “anti-angiogenic agent” as used herein is meant a compound thatblocks, or interferes to some degree, the development of blood vessels.The anti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. The preferred anti-angiogenic factor herein is an antibodythat binds to Vascular Endothelial Growth Factor (VEGF). In an alternateembodiment, the antibody or Fc fusion is administered with a therapeuticagent that induces or enhances adaptive immune response, for example anantibody that targets CTLA-4. In an alternate embodiment, the antibodyor Fc fusion is administered with a tyrosine kinase inhibitor. By“tyrosine kinase inhibitor” as used herein is meant a molecule thatinhibits to some extent tyrosine kinase activity of a tyrosine kinase.Examples of such inhibitors include but are not limited to quinazolines,such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines;pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (ST1571, Gleevec®); Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1-C11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA®, ZD1839, AstraZeneca), and OSI-774(Tarceva®, OSI Pharmaceuticals/Genentech).

A variety of linkers find use in the present invention to generatepolypeptides (see definition above) or antibody—orpolypeptides—conjugates (see definition below). By “linker”, “linkersequence”, “spacer”, “tethering sequence” or grammatical equivalentsthereof, herein is meant a molecule or group of molecules (such as amonomer or polymer) that connects two molecules and often serves toplace the two molecules in a preferred configuration. A number ofstrategies may be used to covalently link molecules together. Theseinclude, but are not limited to polypeptide linkages between N- andC-termini of proteins or protein domains, linkage via disulfide bonds,and linkage via chemical cross-linking reagents. In one aspect of thisembodiment, the linker is a peptide bond, generated by recombinanttechniques or peptide synthesis. Choosing a suitable linker for aspecific case where two polypeptide chains are to be connected dependson various parameters, including but not limited to the nature of thetwo polypeptide chains (e.g., whether they naturally oligomerize), thedistance between the N- and the C-termini to be connected if known,and/or the stability of the linker towards proteolysis and oxidation.Furthermore, the linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 30 amino acid residues.Preferably, the linker is from about 1 to 30 amino acids in length, withlinkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1819 and 20 amino acids in length being preferred. In addition, the aminoacid residues selected for inclusion in the linker peptide shouldexhibit properties that do not interfere significantly with the activityof the polypeptide. Thus, the linker peptide on the whole should notexhibit a charge that would be inconsistent with the activity of thepolypeptide, or interfere with internal folding, or form bonds or otherinteractions with amino acid residues in one or more of the monomersthat would seriously impede the binding of receptor monomer domains.Useful linkers include glycine-serine polymers (including, for example,(GS)n, (GSGGS)n, (GGGGS)n and (GGGS)n, where n is an integer of at leastone), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers such as the tether for the shaker potassium channel,and a large variety of other flexible linkers, as will be appreciated bythose in the art. Glycine-serine polymers are preferred since both ofthese amino acids are relatively unstructured, and therefore may be ableto serve as a neutral tether between components. Secondly, serine ishydrophilic and therefore able to solubilize what could be a globularglycine chain. Third, similar chains have been shown to be effective injoining subunits of recombinant proteins such as single chainantibodies. Suitable linkers may also be identified by screeningdatabases of known three-dimensional structures for naturally occurringmotifs that can bridge the gap between two polypeptide chains. In apreferred embodiment, the linker is not immunogenic when administered ina human patient. Thus linkers may be chosen such that they have lowimmunogenicity or are thought to have low immunogenicity. For example, alinker may be chosen that exists naturally in a human. In a preferredembodiment the linker has the sequence of the hinge region of anantibody, that is the sequence that links the antibody Fab and Fcregions; alternatively the linker has a sequence that comprises part ofthe hinge region, or a sequence that is substantially similar to thehinge region of an antibody. Another way of obtaining a suitable linkeris by optimizing a simple linker, e.g., (Gly4Ser)n, through randommutagenesis. Alternatively, once a suitable polypeptide linker isdefined, additional linker polypeptides can be created to select aminoacids that more optimally interact with the domains being linked. Othertypes of linkers that may be used in the present invention includeartificial polypeptide linkers and inteins. In another embodiment,disulfide bonds are designed to link the two molecules. In anotherembodiment, linkers are chemical cross-linking agents. For example, avariety of bifunctional protein coupling agents may be used, includingbut not limited to N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1971, Science 238:1098.Chemical linkers may enable chelation of an isotope. For example,Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see PCT WO 94/11026).The linker may be cleavable, facilitating release of the cytotoxic drugin the cell. For example, an acid-labile linker, peptidase-sensitivelinker, dimethyl linker or disulfide-containing linker (Chari et al.,1992, Cancer Research 52: 127-131) may be used. Alternatively, a varietyof nonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use to link the Fc variants described herein to afusion partner to generate an Fc fusion, or to link the antibodies andpolypeptides described herein to a conjugate.

In one embodiment, an antibody or polypeptide described herein isconjugated or operably linked to another therapeutic compound, referredto herein as a conjugate. The conjugate may be a cytotoxic agent, achemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosinekinase inhibitor, a toxin, a radioisotope, or other therapeuticallyactive agent. Chemotherapeutic agents, cytokines, anti-angiogenicagents, tyrosine kinase inhibitors, and other therapeutic agents havebeen described above, and all of these aforemention therapeutic agentsmay find use as antibody or Fc fusion conjugates. In an alternateembodiment, the antibody or Fc fusion is conjugated or operably linkedto a toxin, including but not limited to small molecule toxins andenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Small moleculetoxins include but are not limited to calicheamicin, maytansine (U.S.Pat. No. 5,208,020), trichothene, and CC1065. In one embodiment of theinvention, the antibody or polypeptide is conjugated to one or moremaytansine molecules (e.g. about 1 to about 10 maytansine molecules perantibody molecule). Maytansine may, for example, be converted toMay-SS-Me which may be reduced to May-SH3 and reacted with modifiedantibody or Fc fusion (Chari et al., 1992, Cancer Research 52: 127-131)to generate a maytansinoid-antibody or maytansinoid-Fc fusion conjugate.Another conjugate of interest comprises an antibody or Fc fusionconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Dolastatin 10 analogs such asauristatin E (AE) and monomethylauristatin E (MMAE) may find use asconjugates for the Fc variants described herein (Doronina et al., 2003,Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood102(4):1458-65). Useful enzymatically active toxins include but are notlimited to diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, PCT WO 93/21232. Thepresent invention further contemplates a conjugate or fusion formedbetween an antibody or Fc fusion described herein and a compound withnucleolytic activity, for example a ribonuclease or DNA endonucleasesuch as a deoxyribonuclease (DNase).

In an alternate embodiment, an Fc variant protein described herein isconjugated or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies and Fc fusions. Examplesinclude, but are not limited to, At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², and radioactive isotopes of Lu.

In yet another embodiment, an Fc varaint described herein is conjugatedto a “receptor” (such streptavidin) for utilization in tumorpretargeting wherein the antibody-receptor or Fc fusion-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” (e.g. avidin) which is conjugated to acytotoxic agent (e.g. a radionucleotide). In an alternate embodiment,the antibody or Fc fusion is conjugated or operably linked to an enzymein order to employ Antibody Dependent Enzyme Mediated Prodrug Therapy(ADEPT). ADEPT may be used by conjugating or operably linking theantibody or Fc fusion to a prodrug-activating enzyme that converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO 81/01145) toan active anti-cancer drug. See, for example, PCT WO 88/07378 and U.S.Pat. No. 4,975,278. The enzyme component of the immunoconjugate usefulfor ADEPT includes any enzyme capable of acting on a prodrug in such away so as to covert it into its more active, cytotoxic form. Enzymesthat are useful in the method of this invention include but are notlimited to alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as .beta.-galactosidase andneuramimidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized with.alpha.-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458).Antibody-abzyme and Fc fusion-abzyme conjugates can be prepared fordelivery of the abzyme to a tumor cell population.

Other modifications of the Fc Variants comprise linking said protein orpoly-peptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol.

Pharmaceutical compositions are encompassed wherein a Fc variant proteinor polypeptide or Fc fusion described herein and one or moretherapeutically active agents are formulated. Formulations of theantibodies and Fc fusions described herein are prepared for storage bymixing said antibody or Fc fusion having the desired degree of puritywith optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed., 1980), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, acetate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, corn and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® orpolyethylene glycol (PEG). In a preferred embodiment, the pharmaceuticalcomposition that comprises the antibody or Fc fusion described herein isin a water-soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferrably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

In certain embodiments, the proteins and polypeptides disclosed hereinare formulated as immunoliposomes. A liposome is a small vesiclecomprising various types of lipids, phospholipids and/or surfactant thatis useful for delivery of a therapeutic agent to a mammal. Liposomescontaining the antibody or Fc fusion are prepared by methods known inthe art, such as described in Epstein et al., 1985, Proc Natl Acad SciUSA, 82:3688; Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S.Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. A chemotherapeutic agent or other therapeutically active agentis optionally contained within the liposome (Gabizon et al., 1989, JNational Cancer Inst 81:1484).

In certain embodiments, the antibodies, polypeptides, and othertherapeutically active agents are entrapped in microcapsules prepared bymethods including but not limited to coacervation techniques,interfacial polymerization (for example using hydroxymethylcellulose orgelatin-microcapsules, or poly-(methylmethacylate) microcapsules),colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules), andmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymer, which matrices are in the form of shaped articles,e.g. films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOTS (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease®) (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-glycolide(PLG).

In embodiments, the concentration of the therapeutically active antibodyor polypeptide described herein in the formulation varies from about 0.1to 100 weight %. In one embodiment, the concentration of the antibody orpolypeptide is in the range of 0.003 to 1.0 molar. In order to treat apatient, a therapeutically effective dose of the antibody or polypeptidedescribed herein may be administered. By “therapeutically effectivedose” herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.01 to 100 mg/kg of bodyweight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight,with 1 to 10 mg/kg being preferred. As is known in the art, adjustmentsfor antibody or polypeptide degradation, systemic versus localizeddelivery, and rate of new protease synthesis, as well as the age, bodyweight, general health, gender, diet, time of administration, druginteraction and the severity of the condition may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

Administration of the pharmaceutical composition comprising a protein orpolypeptide described herein in the form of a sterile aqueous solution,is done in a variety of ways, including, but not limited to orally,subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx®inhalable technology commercially available from Aradigm, or Inhance®pulmonary delivery system commercially available from InhaleTherapeutics), vaginally, parenterally, rectally, or intraocularly. Insome instances, for example for the treatment of wounds, inflammation,etc., the antibody or Polypeptide may be directly applied as a solutionor spray. As is known in the art, the pharmaceutical composition may beformulated accordingly depending upon the manner of introduction.

According to the teachings herein, a polypeptide can be prepared with avariant Fc region which has improved, or diminished, ADCC activity. Suchmolecules find applications in the treatment of different disorders.

In certain embodiments, the polypeptide variant with improved ADCCactivity is employed in the treatment of diseases or disorders wheredestruction or elimination of tissue or foreign micro-organisms isdesired. For example, the polypeptide may be used to treat cancer;inflammatory disorders; infections (e.g. bacterial, viral, fungal oryeast infections); and other conditions (such as goiter) where removalof tissue is desired, etc.

Where the polypeptide variant has diminished ADCC activity, suchvariants are used to treat diseases or disorders where a Fcregion-containing polypeptide with long half-life is desired, but thepolypeptide preferably does not have undesirable effector function(s).For example, in an embodiment, the Fc region-containing polypeptide isan anti-tissue factor (TF) antibody; anti-IgE antibody; andanti-integrin antibody (e.g. an anti-aα4β7 antibody). The desiredmechanism of action of such Fc region-containing polypeptides may be toblock ligand-receptor binding pairs. Moreover, the Fc-region containingpolypeptide with diminished ADCC activity may be an agonist antibody.

In an aspect are provided methods for generating Fc proteins that aresubsequently screened experimentally to single out optimized Fcproteins. General methods for antibody molecular biology, expression,purification, and screening are described in Antibodies: A LaboratoryManual by Harlow & Lane, New York: Cold Spring Harbor Laboratory Press,1988.

One embodiment described herein relates to a rational design process toidentify Fc proteins with improved selectivity and binding affinity tothe Fc receptors. In one aspect, the invention includes understandingthe structural features associated with dynamic properties which drivethe interaction between the Fc and its receptors. Another embodimentdescribed herein provides a method for identifying Fc proteinpolypeptides based on their binding affinities to FcγRIIa, FcγRIIb, andFcγRIIIa, electrostatics, solvation, packing, packing density, hydrogenbinding networks and entropic effects associated with the dynamic natureof the protein molecule. The method of the current invention furtherprovides for constructing the in silico identified mutants by, forexample site-directed mutagenesis and/or de novo synthesis, andexpression in mammalian cells for in vitro validation.

In one embodiment described herein, the Fc protein polypeptidesidentified in silico are reverse engineered to create nucleic acids thatencode the member sequences, that may then be cloned into host cells,expressed and assayed, if desired. These practices are carried out usingwell-known procedures. For example, a variety of methods that may finduse in the present invention are described in Molecular Cloning—ALaboratory Manual, 3rd Ed. (Maniatis, Cold Spring Harbor LaboratoryPress, New York, 2001).

Fc proteins are screened using a variety of methods, including but notlimited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label.

Design Strategies

A rational design process was undertaken to design Fc proteins withmultiple amino acid substitutions that synergistically provide enhancedselectivity and binding affinity to a target Fc receptor. Central tothis process is the understanding of structural features and associateddynamic properties which drive the interaction between the Fc and itsreceptors. Starting with the known co-complex structure between the Fcand the FcγRIIIb, homology models were constructed for the receptors ofquestion: FcγRIIa, FcγRIIb, and FcγRIIIa. Applying a proprietary suiteof in silico structure and dynamics-based technologies (including butnot limited to ZymeCAD™ and ResidueNetworks™ (as described in WO2009/098596, US20070276791 and US 20080147360)), unique insights intothe structure-function relationships of these protein-proteininteractions were identified and led to the proposal of uniquecombinations of mutations which were predicted to preferentially bind tovarious Fc receptors, and in certain examples, with improved bindingstrength. The primary quantitative metric to evaluate the Fc proteins isthe binding energies. However, a number of other parameters such aschanges in electrostatics, solvation, packing and packing density,hydrogen binding networks and entropic effects associated with thedynamic nature of the protein molecule are also employed in selectingattractive Fc proteins.

WO 2009/098596 provides methods and systems of determining biopolymerprofiles and correlations between structural units (residues) of abiopolymer based on sampling of the conformational space available tothe molecule. The correlations between these structural units arefurther used to find coupled residue networks in the protein.

In one embodiment, the functional and/or biophysical properties of Fcproteins are screened in an in vitro assay. Assays may employ a varietyof detection methods including but not limited to chromogenic,fluorescent, luminescent, or isotopic labels. Assay to detect ADCCinclude but are not limited to, ADCC reporter assays, cytotoxicityassays, chromium release assay, europium release assay, sulfur releaseassay, and flow cytometry. Examples of well known assays can be found inthe art, for example in Stavenhagen et al. (2007) “Fc Optimization ofTherapeutic Antibodies Enhances Their Ability to Kill Tumor Cells Invitro and Controls Tumor Expansion In vivo via low-affinity ActivatingFcγ Receptors,” Cancer Res. 67:882 and Stavenhagen et al. (2008)“Enhancing the potency of therapeutic monoclonal antibodies via Fcoptimization,” Adv. Enzyme Regul. 48:152.

The biological properties of the antibodies and polypeptides thatcomprise the Fc proteins described herein may be characterized in cell,tissue, and whole organism experiments. Drugs are often tested inanimals, including but not limited to mice, rats, rabbits, dogs, cats,pigs, and monkeys, in order to measure a drug's efficacy for treatmentagainst a disease or disease model, or to measure a drug'spharmacokinetics, toxicity, and other properties. Therapeutics are oftentested in mice, including but not limited to nude mice, SCID mice,xenograft mice, and transgenic mice (including knock-ins and knockouts).

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of this invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

Example 1 Illustration of Rational Design of Polypeptides Having aCombination of Mutations Selected for Producing Selected FcγR BindingProfile

Antibodies Targeting the HER2/Neu Receptor:

The Human Epidermal growth factor Receptors (HERs) are proteins embeddedin the cell membrane and communicate molecular signals from outside thecell to inside the cell, and turn genes on and off. The HER proteinsregulate cell growth, survival, adhesion, migration, anddifferentiation—functions that are amplified or weakened in cancercells. Human Epidermal growth factor Receptor 2″—a member of theepidermal growth factor receptor family, is a protein giving higheraggressiveness in breast cancers. HER2/neu has also been designated asCD340. Approximately 15-20 percent of breast cancers have anamplification of the HER2/neu gene or overexpression of its proteinproduct. Overexpression of this receptor in breast cancer is associatedwith increased disease recurrence and worse prognosis. Because of itsprognostic role as well as its ability to predict response totrastuzumab, breast tumors are routinely checked for overexpression ofHER2/neu. Overexpression also occurs in other cancer such as ovariancancer, stomach cancer, and biologically aggressive forms of uterinecancer, such as uterine serous endometrial carcinoma.

Trastuzumab is a humanized monoclonal antibody that binds to the domainIV of the extracellular segment of the HER2/neu receptor. Cells treatedwith trastuzumab undergo arrest during the G1 phase of the cell cycle sothere is reduced proliferation. It has been suggested that trastuzumabinduces some of its effect by downregulation of HER2/neu leading todisruption of receptor dimerization and signaling through the downstreamPI3K cascade. P27Kip1 is then not phosphorylated and is able to enterthe nucleus and inhibit cdk2 activity, causing cell cycle arrest. Also,trastuzumab suppresses angiogenesis by both induction of antiangiogenicfactors and repression of proangiogenic factors. It is thought that acontribution to the unregulated growth observed in cancer could be dueto proteolytic cleavage of HER2/neu that results in the release of theextracellular domain. Trastuzumab has been shown to inhibit HER2/neuectodomain cleavage in breast cancer cells.

Design of a Polypeptide Comprising a Combination of Modifications thatProduce a Selected FcγR Binding Profile:

Some of the proteins and polypeptides disclosed herein are designedbased on the impact of selected amino acid substitutions to the proteininteractions and dynamics, which are indicative of enthalpic andentropic changes, respectively. Relative changes to protein bindingcharacteristics, especially compared to the wild-type system, can beoptimized.

In this example, a polypeptide is designed with the goal of selectingamino acid mutations that increase binding selectivity to the FcγRIIIareceptor when compared to a wild-type antibody (Trastuzumab/Herceptin®).The rational design performed resulted in a quadruple variant(S239E/S298A/K326A/A327H). In the following example, the main strategiesof introducing FcγRIIIa-specific electrostatic interactions and addingsteric repulsion to both FcγRIIa and FcγRIIb receptors are highlightedsuch that the selectivity design goal was achieved. In some cases, themutations' contribution to binding on one chain of the Fc is differentthan the other chain and these chain-specific effects are alsodemonstrated. The specific effects to binding as a result of each of thefour mutations are discussed with the binding profile of the Fc variantbeing an overall synergistic combination of the structural and dynamicchanges.

A327H as a Steric Selectivity Driver:

His327 is a much bulkier group compared to the wild-type alanine andthis substitution is a main selectivity driver that has minimal impacton the FcγRIIIa receptor while being detrimental for FcγRIIa andFcγRIIb. As shown in FIG. 1, the binding interface and theprotein-protein interactions with the FcγRIIIa are preserved with theaddition of His327 when compared to the wild-type Fc, which suggeststhat binding will not be affected between these two partners. Theprimary factor for the interface stabilization is due to the presence ofthe sidechain hydroxyl group from Tyr132 in FcγRIIIa. This non-conservedmoiety is not present in FcγRIIa and hence the addition of His327resulted in a major destabilization of the binding interface, mostnotably with the rapid dynamics observed for the receptor's neighboringHis134 residue (FIG. 1). It is anticipated that binding to the IIareceptor will be severely disrupted.

While FcγRIIIa and FcγRIIa share a conserved His134 residue that isdifferentially affected with the addition of His327 to the Fc, FcγRIIbcontains of an even bulkier Arg134. While this residue is also perturbedwith the His134 mutation, it is however able to adopt a pseudo-favorableconformation that is stabilized with a serine sidechain on the Fc (FIG.1). It is expected that binding of the Fc to the IIb receptor will bereduced but not abolished because of the stabilized Arg rotomer.

S239E as an Electrostatic General Stabilizer and a Selectivity Driver:

The mutation S239E displays Fc chain-specific interactions to thereceptors thereby facilitating both of its roles as a generalstabilizing mutation as well as a selectivity driver for the FcγRIIIareceptor.Glu239 on chain-B of the Fc (FIG. 1) is a general stabilizingresidue across all three receptors as it is able to form a hydrogen bondwith conserved Lys120 residues. General stabilizing residues arebeneficial in protein variant design as they ensure that the integrityof the binding interfaces is maintained when numerous mutations areintroduced within confined areas.

The S239E mutation, when introduced on chain-A of the Fc (FIG. 2) is aselectivity driver for improved FcγRIIIa binding as it is able to form abifurcated electrostatic interaction with the non-conserved Lys161 whilethe enhanced binding interaction is not possible with the Thr161 in theFcγRIIa and IIb receptors.

S298A as Both a Steric and Electrostatic Selectivity Driver

The Ser298 residue in the wild-type antibody Fc interacts with Ser129 inthe FcγRIIa and IIb receptors via hydrogen bonding while the receptors'Lys128 is stabilized intramolecularly and is not involved in any bindinginteractions (FIG. 3). The S298A mutation removes one of the keyelectrostatic binding partners between the Fc and the receptors and istherefore expected to drastically reduce binding to FcγRIIa and IIbreceptors.

The wild-type Fc binds to the FcγRIIIa receptor using a differentmechanism, with the interaction mediated by the backbone carbonyl groupof Ser298 on the Fc with Lys128 from the receptor, which is able topartially occupy the binding interface. The sidechain flexibility andmotions required for this interaction was demonstrated using moleculardynamics simulations (FIG. 3). The FcγRIIIa's Lys128 is ‘locked’ andpre-positioned in the binding conformation when the S298A mutation isintroduced in the Fc.

Synergistic Combination of Mutations

By combining the above computational and structural insights in the Fcdesign process; the present disclosure provides a new variant that has,relative to the wild-type Fc, improved FcγRIIIa binding while havingreduced binding to FcγRIIb and substantially abolished binding toFcγRIIa. As illustrated by in vitro binding assays with the resultingbinding profile shown in FIG. 4.

These results also demonstrate the unexpected synergistic nature ofthese mutations (FIG. 4). With the single S239E mutation, it is shownthat the variants cannot clearly differentiate binding between thehighly homologous receptors. With the addition of the S298A/K326A/A327Hmutation combination, FcγRIIIa selectivity was improved.

Example 2 In Vitro and Ex Vivo Validation of Designed Antibodies

Once designed in silico individual antibodies are tested according tothe methods described in Stavenhagen et al. (2007) Cancer Res. 67:882and Stavenhagen et al. (2008), Adv. Enzyme Regal., 48:152.

Briefly, the gene for each mutant is constructed by standard chemicalsynthesis using, for example, Trastuzumab IgG1 as the wild-typeframework. After cloning into a suitable vector, the mutant Fcpolypeptides are expressed in mammalian HEK293 cells. The FcγRIIa,FcγRIIb and FcγRIIIa are also cloned and expressed in HEK293 cells. Thebinding affinities of the antibodies to each of the three receptors isthen determined by surface plasmon resonance.

Surface Plasmon Resonance Analysis:

Affinity of Fcγ receptors to antibody Fc was measure by SPR (surfacePlasmon resonance) using a ProteOn XPR36 system from BIO-RAD. HER-2 inbuffer (10 mM Hepes pH 6.8) was immobilized on CM5 chip through aminecoupling until 3000 RU. Fc variants in an antibody format containinganti HER2 F(ab)2 were immobilized to the HER-2 surface to 300 RU.Running buffer and the surfactant was maintained at pH 6.8. Purifiedanalyte FcR was diluted in its running buffer and injected at a flowrate of 20-30 mul/min for 2 minutes, followed by dissociation foranother 4 minutes. Five twofold dilutions of each antibody beginning at20 nM were analyzed in triplicate. Sensograms were fit globally to a 1:1Langmuir binding model. All experiments were conducted at roomtemperature. The in vitro binding Kd determined by SPR for each variantis shown in Table 1.

Antibody-Dependent Cellular Cytotoxicity Analysis:

The SKBR-3 cells were used as target cells in these experiments. A freshvial of cryopreserved SKBR3 cells was thawed and a robust cultureestablished. The SKBR3 cells were maintained in McKoy's medium enrichedwith 10% Fetal Bovine Serum and 1% PenStrep. The cells were passagedregularly upon attaining ˜75% confluence (approximately every 3-4 days).To verify the binding of the variant antibodies to the target cells(SKBR3), binding curves for all variants and positive control(Herceptin) were obtained. SKBR-3 cells were washed with PBS andresuspended in FACS (Fluorescence-activated cell sorting) tubes at 2×10⁵cells per tube in 100 μl volume of PBS/1% BSA. Antibodies were added tothe tubes to achieve 0.1, 1 and 10 μg/ml final concentration, cellsincubated on ice for 1 hour, washed with 1% BSA/PBS and resuspended in1:200 dilution of FITC-conjugated anti-human IgG. Cells were incubatedon ice for another 40 min, washed again and resuspended in 200 μl ofPBS. 10 μl of 10 mg/ml Propidium Iodide were added to each tube, andsamples were analyzed by FACS. FACS gates were set to exclude dead cells(PI+). The Mean Fluorescence Intensity (“MFI”) for each sample wasmeasured. Cells stained with secondary but not primary antibody wereused as negative control. As measured from the fold-difference in meanfluorescence intensity (“MFI”), all variants demonstrated levels ofbinding comparable to Herceptin.

To establish an effector:target cell (E:T) ratio and duration ofincubation to achieve suitable cell killing, a preliminary ADCCexperiment was performed using Herceptin as a positive control. SKBR-3cells were seeded in flat bottom 96 well plates a day prior toexperiment, at 2×10⁴ cells per well in 200 μl of culture media. PBMCswere purified from fresh buffy coats from five different donors usingFicoll gradient centrifugation, washed 3 times with PBS and resuspendedin RPMI containing 10% Heat Inactivated FBS and 10 ng/ml IL-2. Twentyfour hours later, three wells containing SKBR-3 cells were trypsinizedto verify cell count and to determine the exact number of PBMCs requiredto achieve the desired E:T ratio. Target cells were labeled with 10μg/ml CFSE immediately prior of assay. Antibody (Herceptin) was added tocells at 1 and 10 μg/ml and incubated for 15 min. PBMCs at 10:1, 50:1and 100:1 effector:target (E:T) ratio were then added to correspondingwells, and plates were briefly spun down at low RPM to concentrate cellsin the bottom of the wells. The plates were then incubated in a standardtissue culture incubator for four, eight and twenty four hours.Following treatment, cells were harvested and added to 400 μl PBScontaining propidium iodide (“PI”), a viability stain, and immediatelyanalyzed by FACS. The extent of ADCC activity was determined bymeasuring the frequency of PI⁺ green cells (killed targets) as thefraction of total target cells (PI⁻ and PI⁺ green cells). The pilot ADCCexperiment demonstrated significant ADCC activity of Herceptin in SKBR3cells. At 24 hours, higher E:T ratios (50:1 and 100:1) appeared toresult in some antibody independent cell death. Because the killing ofthe cells at antibody concentrations of 1 and 10 μg/ml isindistinguishable, ADCC activity appears to be saturating above 1 μg/ml.Optimal results were observed at 24 hours with an E:T ratio of 10:1.

For the subsequent ADCC experiment, PBMCs from five buffy coats werepurified using Ficoll gradient centrifugation. Following centrifugationand washes, cells were resuspended in 100 ml of pre-warmed RPMI. Thecells were counted, and viability was determined by the Trypan Blueexclusion. An aliquot of cells from each donor was cryopreseved forfuture use. An additional aliquot of cells was used for FACS-basedgenotyping for the 158V/F CD16 polymorphism using a two-antibodystaining protocol that exploits the fact that the G38 anti-CD16 antibodybinds with equal affinity to both V and F alleles and comparing thatwith staining using the MEM-154 anti-CD16 antibody that has a loweraffinity for the V allele than for the F allele (S. Bottcher et al.,2005, Journal of Immunological Methods, Volume 306, Issues 1-2, pp128-136). Based on the CD16 genotyping results, samples from threedonors heterozygous in the F/V, high cell viability and no endogenouscell killing were selected. All data points were obtained in triplicatewells and the viability for each donor was normalized to the viabilityof SKBR-3 cells incubated with PBMCs of the same donor, but without testantibody. The average ADCC results for these samples are summarized inTable 1.

As seen from table 1, the ex vivo ADCC assay revealed that a designedvariants had significantly lowered ADCC than the wild-type antibody.This shows that the variations described herein are useful in modulatingADCC.

TABLE 1 Results from SPR and ADCC assay for different designedantibodies. ex vivo in vitro Binding Kd [M] in silico ΔΔG solv[kcal/mol] ADCC assay IIIa (F) IIa (H) IIb IIIa (F) IIa (H) IIb RelativeEC50 Mutations Compared to Wildtype Trastuzumab variant variant variantvariant variant variant [ug/mL] Wildtype Trastuzumab 1.00E−06 4.62E−075.41E−07 +++ +++ +++ 1.0 G237F S239E A327H — — 1.58E−06 8.19E−061.16E−06 ++++ +++ ++ 0.7 G237F A327L A330I — — NB NB 1.46E−06 ++ ++++++++ 0.0 S239E A327L A330I — — 1.47E−06 NB 6.15E−06 +++++ ++ + ND L235AS239E D265E A327H — NB NB NB +++++ +++++ + 0.0 S239E S267E H268D — —1.75E−07 6.42E−07 1.61E−08 +++++ +++++ +++++ 2.1 G237F S239E D270N — —NB NB NB ++ + + 1.0 G236E G237F S239E — — ND NB 8.56E−07 +++ + + NDS239E D265S H268D I332E — ND 4.44E−06 4.16E−06 ++++ + + ND S239E D265SI332E — — 2.46E−06 NB NB ++++ +++ + ND G237F S239E D265E — — NB NB2.95E−06 +++++ +++++ + ND G237F S239E H268D — — 5.94E−07 7.40E−076.84E−08 +++ +++++ +++++ 0.7 S239D D265S S298A I332E 1.04E−06 NB4.01E−06 +++ ++ +++++ ND S239E S298A K326A A327H — 3.33E−07 NB 2.71E−06+++++ ++ ++ ND G237F S298A A330L I332E — 4.29E−07 NB 7.63E−06 +++ +++++++ 5.3 G236E D270N A327V I332E — NB NB NB +++++ + + ND H268E D270ES267G — — 1.27E−06 5.31E−07 5.83E−07 +++++ +++++ +++++ ND H268D K326AA327H — — 1.33E−06 1.10E−06 2.71E−07 ++++ ++++ +++++ ND H268D E269LS298A K326A A327H 2.43E−06 NB 6.17E−06 + ++ +++++ ND D265E S267D A330S —— NB NB 1.59E−06 + +++++ ++++ 0.0 +++++ <−5 kcal/mol; ++++ −5 to −2kcal/mol; +++ Between −2 and 2 kcal/mol; ++ 2 to 5 kcal/mol; + >5kcal/mol NB: no binding; ND: Not Determined. Relative EC50: >1 =Enhanced ADCC; <1 = Supressed ADCC; ND = Not Determined

Example 3

Additional antibodies comprising modifications based on the in silicomethods described above are summarized in Table 2.

TABLE 2 in silico ΔΔG solv [kcal/mol] IIIa (F) IIIa (V) IIa (H) IIa (R)IIb (F) IIb (Y) Mutations variant variant variant variant variantvariant WT Herceptin — — — — — +++ +++ +++ +++ +++ +++ D270L Y300L A330K— — + + +++++ ++++ ++++ +++ G236A S239D D270L I332E — +++++ +++++ ++++++ +++++ +++++ G237F S267G N325F — — + +++ +++ + ++ + G237F V266L S267D— — ++ ++ +++ ++++ +++++ ++++ L234F S267G N325L — — ++ + + + +++ +++L234F S267E N325L — — +++ + + + +++++ ++++ +++++ <−5 kcal/mol; ++++ −5to −2 kcal/mol; +++ Between −2 and 2 kcal/mol; ++ 2 to 5 kcal/mol; + >5kcal/mol NB: no binding; ND: Not Determined

Example 4 Treatment of Non-Hodgkins Lymphoma Using Anti-CD20 Antibodies

An antibody comprising a combination of modifications as describedherein is administered to a patient having low-grade or follicular NHL.The recommended dosage for patients with low-grade or follicular NHL is375 mg/m 2 infused i.v. at weekly intervals for a total of four doses.In a majority of patients, this can be accomplished in an outpatientclinic over a 22-day period.

Just before administration, the antibody preparation is diluted with 5%dextrose in water or 0.9% sodium chloride injection to a finalconcentration of 1-4 mg/mL. A 1-mg/mL dilution is preferable tofacilitate adjustments in the infusion and to avoid adverse effectspotentially caused by inadvertently rapid administration. Preparedinfusions are stable in polyvinyl chloride or polyethylene bags at 2-8°C. (36-46° F.) for 24 hours and at room temperature for an additional 12hours. Unused portions of undiluted drug must be discarded because ofthe absence of a preservative.

Thirty to 60 minutes before each infusion, acetaminophen 650-1000 mg anddiphenhydramine hydrochloride 50-100 mg can be administered to helpprevent infusion-related effects. Infusion may be through either acentral or peripheral i.v. catheter but should never be given by i.v.push or bolus injection, owing to the risk of potentially seriousinfusion-related adverse effects. Before beginning each infusion, theadministering clinician must prime the i.v. tubing with drug-containingsolution to guarantee that active drug rather than other solution isbeing infused from the beginning. The first infusion should be initiatedat 50 mg/hr with the rate increased by 50 mg/hr every 30 minutes astolerated until a maximum rate of 400 mg/hr is reached. Subsequentinfusions may be started at 100 mg/hr, with 100-mg/hr increases every 30minutes as tolerated until the maximum rate (400 mg/hr) is attained. Inpatients with a large tumor burden (white blood cell count, >25,000/mm3), an initial infusion rate of 25 mg/hr should be considered.

Example 5 Treatment of Systemic Lupus Erythematosus inSerologically-Active Patients

All subjects receive anti-CD22 antibodies incorporating the Fcpolypeptide modifications described herein monthly, with loading doseson days 8 and 15 of month one, until disease progression or subjectdiscontinuation. The drug is prepared in a 10 mg/ml prepared in 17.5vials. Administration is carried out by slow intravenous infusion usingPBS as a vehicle/buffer for the infusion procedures. All patients weregiven 1200 mg antibody given in 2 doses every other week in 12 weektreatment cycles. Assessments of the patients treated were accomplishedthrough a combined response index analysis evaluating BILAG, SLEDAI, anda physician's global assessment and treatment failure status.

Example 6 Treatment of BLL Using Anti-CD22 Antibodies

The dosage used is 120-1000-mg/m2 by infusion with diffuse large B-celllymphoma.

Sub-Cutaneous Administration of Antibodies comprising the amino acidmodifications described herein

The subcutaneous immunoglobulin is infused in the subcutaneous tissue onthe abdomen twice or thrice a week, with a maximal speed of 2 mL/h.Every time 20 mL is infused, the needle is removed to a new place.

Example 7 Administration of Immunoglobulin to Control Chronic DiseaseSuch as Type B Hepatitis

Continuous monthly administration of anti-hepatitis B antibodiesincorporating the Fc polypeptides described herein at 5000 IU a month asa treatment results in reduced serum levels of the patient's ownanti-hepatitis B antibodies and decreased symptoms of liver disease.

Example 8 Treatment of Organ Rejection Using Anti-CD20 Antibodies

Treatment of organ transplant patients is done either weekly, biweekly,bi-monthly, or monthly, by infusion of anti-CD20 antibodies comprisingthe amino acid modifications provided herein from 100 to 1000 mg/m2,according the the above protocol for infusion and pretreatment.

The polypeptides and methods disclosed herein are used, withoutlimitation to develop antibodies and polypeptides based on synergisticimprovements to the Fc region of the following antibodies: Bevacizumab(Avastin); Abciximab (ReoPro); Adalimumab (Humira); Alemtuzumab(Campath); Cetuximab (Erbitux); Efalizumab (Raptiva); Etanercept(Enbrel); Gemtuzumab oxogamicin (Mylotarg); Infliximab (Remicade);Natalizumab (Tysabri, aka Antegren); Omalizumab (Xolair); Palivizumab(Syangis); Rituximab (Rituxan); Trastuzumab (Herceptin); Golimumab(Simponi); Panitumumab (Vectibix); Canakinumab (ILARIS); Ustekinumab(Stelara); Denosumab (Prolia); Ofatumumab (Arzerra)

While specific embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. It isintended that the following claims define the scope of the invention andthat methods and structures within the scope of these claims and theirequivalents be covered thereby.

What is claimed is:
 1. A polypeptide comprising a variant Fc region, wherein said variant Fc region comprises three or more amino acid modifications relative to a wild-type Fc region, and has an altered effect relative to a polypeptide comprising a wild-type Fc region or variant Fc region having only one of the three or more amino acid modifications; wherein at least two of the three or more modifications provide a synergistic effect compared to single position modifications at the at least two positions thereby exhibiting a selected binding profile to Fcγ receptors.
 2. The polypeptide of claim 1 wherein the amino acid modifications produce amino acid interactions and dynamics that result in enhanced binding affinity and/or specificity to a first Fcγ receptor while diminishing binding affinity and/or specificity to a second Fcγ receptor compared to a polypeptide that lacks the at least three or more amino acid modifications.
 3. The polypeptide of claim 2 wherein the first Fcγ receptor is FcγRIIIa receptor and the second Fcγ receptor is FcγRIIa or FcγRIIb.
 4. The polypeptide of claim 1 wherein the amino acid modifications produce favorable FcγRIIIa-specific interactions and/or unfavorable interactions with FcγRIIa and/or FcγRIIb receptors.
 5. The polypeptide of claim 1 wherein the amino acid modifications have minimal impact on the FcγRIIIa receptor while producing detrimental effects on binding of the polypeptide to FcγRIIa and/or FcγRIIb.
 6. The polypeptide of claim 1 wherein the polypeptide comprises modifications S239E/D265S/I332E.
 7. The polypeptide of claim 1 wherein the polypeptide comprises modifications H268D/E269L/S298A/K326A/A327H.
 8. The polypeptide of claim 2 wherein the first Fcγ receptor is FcγRIIa receptor and the second Fcγ receptor is FcγRIIIa or FcγRIIb.
 9. The polypeptide of claim 1 wherein the amino acid modifications produce favorable FcγRIIa-specific interactions and/or unfavorable interactions with FcγRIIIa and/or FcγRIIb receptors.
 10. The polypeptide of claim 1 wherein the amino acid modifications have minimal impact on the FcγRIIa receptor while producing detrimental effects on binding of the polypeptide to FcγRIIIa and/or FcγRIIb.
 11. The polypeptide of claim 2 wherein the first Fcγ receptor is FcγRIIb receptor and the second Fcγ receptor is FcγRIIIa receptor or FcγRIIa receptor.
 12. The polypeptide of claim 1 wherein the amino acid modifications produce favorable FcγRIIb-specific interactions and/or unfavorable interactions with FcγRIIIa and/or FcγRIIa receptors.
 13. The polypeptide of claim 1 wherein the amino acid modifications have minimal impact on the FcγRIIb receptor while producing detrimental effects on binding of the polypeptide to FcγRIIIa and/or FcγRIIa.
 14. The polypeptide of claim 1 wherein the polypeptide comprises modifications D265E/S267D/A330S.
 15. The polypeptide of claim 1 wherein the polypeptide comprises modifications G237F/A327L/A330I.
 16. The polypeptide of claim 1 wherein the polypeptide comprises modifications G237F/S239E/H268D.
 17. The polypeptide of claim 1 wherein the polypeptide comprises modifications S239E/S267E/H268D.
 18. The polypeptide of claim 1 wherein the polypeptide comprises modifications S239E/S298A/K326A/A327H.
 19. The polypeptide of claim 1 wherein the polypeptide comprises modifications G237F/S298A/A330L/I332E.
 20. The polypeptide of claim 1 wherein the polypeptide comprises modifications G237F/S239E/A327H.
 21. The polypeptide of claim 1 wherein the polypeptide comprises modifications L235A/S239E/D265E/A327H.
 22. The polypeptide of claim 1 wherein the polypeptide comprises modifications G237F/S239E/D270N.
 23. The polypeptide of claim 1 wherein binding of the polypeptide comprising a wild-type Fc region to Fcγ receptors is detectable by an in vitro assay.
 24. The polypeptide of claim 1 wherein the three or more modifications comprise the mutation S239E wherein the polypeptide has higher selectivity in binding to the FcγRIIIa receptor compared to a polypeptide that lacks the S239E mutation.
 25. The polypeptide of claim 1 wherein the three or more modifications comprise the mutation S298A wherein the polypeptide has reduced binding affinity to FcγRIIa and FcγRIIb receptors compared to a polypeptide that lacks the S298A mutation.
 26. The polypeptide of claim 1 wherein the polypeptide comprises the amino acid modifications S298A/K326A/A327H wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the S298A/K326A/A327H modifications.
 27. The polypeptide of claim 1 wherein the polypeptide comprises the amino acid modifications S239E/S298A/K326A/A327H wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the S239E/S298A/K326A/A327H modifications.
 28. The polypeptide of claim 1 wherein the polypeptide comprises the amino acid modifications G236A/S239D/D270L/I332E, wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the G236A/S239D/D270L/I332E modifications.
 29. The polypeptide of claim 1 wherein the polypeptide comprises three amino acid modifications, said mutations selected from D270L/Y300L/A330K, G237F/S267G/N325F, G237F/V266L/S267D, L234F/S267G/N325L, and L234F/S267E/N325L.
 30. The polypeptide of claim 1 wherein the polypeptide comprises modifications of at least three amino acids, said modifications selected from G237F/S239E/A327H, G237F/A327L/A330I, S239E/A327L/A330I, S239E/S267E/H268D, G237F/S239E/D270N, G236E/G237F/S239E, S239E/D265S/I332E, G237F/S239E/D265E, G237F/S239E/H268D, H268E/D270E/S267G, H268D/K326A/A327H, D265E/S267D/A330S, L235A/S239E/D265E, A327H/E269L/K236A, G237F/D270Q/S239E, A330V/I332L/K326, and G236S/A327H/A330I.
 31. The polypeptide of claim 1 wherein the polypeptide comprises simultaneous modification of at least four amino acids, said modifications selected from L235A/S239E/D265E/A327H, S239E/D265S/H268D/I332E, S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, and G236E/D270N/A327V/I332E, and G236A/S239D/D270L/I332E.
 32. The polypeptide of claim 1 wherein the polypeptide comprises the amino acid modifications H268D/E269L/S298A/K326A/A327H.
 33. The polypeptide of any of claims 1-32 wherein the polypeptide comprises the antibody trastuzumab or an antigen binding portion thereof.
 34. The polypeptide of claim 1, wherein one or more amino acid modifications are located between positions 234-330 according to the EU index.
 35. The polypeptide of claim 1 wherein at least three amino acid modifications are selected from the group consisting of: L234Q, L234N, L235A, G236E, E236L, E236D, G237F, G237N, S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268D, H268E, E269L, E269L, D270N, D270I, D270E, S298A, K326A, K326D, A327H, A327V, A327L, A327T, A330V, A330L, A330W, A330I, A330S, I332L, I332D, and 1332E.
 36. The polypeptide of claim 1 wherein said variant Fc region comprises a combination of amino acid modifications wherein said combination is selected from the group consisting of: L235A/S239E/D265E; L235A/G237F/D265E; S239E/E269D/A327H; S239E/G237N/A327H; S239E/G237F/A327V; G237F/D270I/S239E; G237F/A327L/S239E; A327H/E269L/K326A; A330V/I332L/S239E; A327T/E269L/K326A; D270N/A327T/K326A; A330V/I332L/S239E; A330W/I332D/S239E; G236E/D265E/A327H/A330I; D270N/S298A/A327V; G236E/D265E/D270N/A327H/A330I; G236E/D270N/A327H/A330I; G236E/D270N/A327V/I332E; G236E/D270N/A327V/G237F; L234N/S239E/A330I/I332E; L234Q/S239E/A330I/I332E; L234Q/S239E/A330I/I332E/S298A; G237F/S239D/D265E/D270N/S298A; G237F/S239E/D270N/A330L/I332E; G237F/S239E/D270N/A330L/I332E/S298A; S239E/G237F/A327H; G237F/A327L/A330I; S239E/A330I/A327L; D265E/S239E/L235A/A327H; S267E/S239E/H268D; G237F/D270N/S239E; S239E/G237F/G236E; 1332E/D265S/S239E/H268D; I332E/D265S/S239E; D265E/S239E/G237F; S239E/H268D/G237F; S298A/D265S/S239D/I332E; S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E; H268E/D270E/S267G; H268D/K326A/A327H; H268D/K326A/A327H/E269L/S298A; and A330S/D265E/S267D.
 37. The polypeptide of claim 1 wherein said variant Fc region comprises a combination of amino acid modifications wherein said combination is selected from the group consisting of S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G.
 38. The polypeptide of claim 1, wherein said variant Fc region comprises amino acid modification H268D and not modification S267E.
 39. The polypeptide of claim 1, wherein the Fc region of the parent polypeptide is a human IgG Fc region.
 40. The polypeptide of claim 39, wherein the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region.
 41. The polypeptide of claim 1 wherein said polypeptide is an antibody.
 42. The antibody of claim 41, wherein said antibody is a monoclonal antibody, a humanized antibody, or a human antibody.
 43. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of claim
 1. 44. A vector comprising the nucleic acid of claim
 43. 45. A method for producing the polypeptide of claim 1, said method comprising: (i) culturing in a medium a host cell comprising a nucleic acid encoding said polypeptide, under conditions suitable for the expression of said polypeptide; and (ii) recovering the polypeptide from said medium.
 46. A therapeutic antibody that specifically binds a cancer target antigen, said therapeutic antibody comprising a variant Fc region of claim
 1. 47. The therapeutic antibody of claim 46, wherein said therapeutic antibody is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab, zalutumumab, and any other antibodies.
 48. The therapeutic antibody of claim 46, wherein said target antigen is selected from the group consisting of a-chain (CD25) of IL-2R, Amyloid beta, anti-EpCAM×anti-CD3, BLyS (or BAFF), CD11a, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F protein of RSV, G250, glycoprotein IIb/IIIa R, HER2, HER2/neu R, Hsp90, IgE antibody, IL-12/IL-23, IL-1b, IL-5, IL-6 receptor, Integrin alpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF alpha, VEGF-A, and other therapeutically advantageous targets.
 49. A method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of a therapeutic antibody of claim
 46. 50. The method of claim 49, wherein said patient is human.
 51. A method of treating immune disorders in a patient having an immune disorder characterized by an immune antigen, said method comprising administering to said patient a therapeutically effective amount of a therapeutic antibody of claim
 46. 52. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptides of claim 1, and a pharmaceutically acceptable carrier.
 53. The polypeptide of claim 1 wherein the polypeptide comprising a variant Fc region is more effective at mediating ADCC relative to wild type.
 54. The polypeptide of claim 53 wherein the polypeptide comprising a variant Fc region is about 1.5 to about 100 fold more effective in mediating ADCC relative to wild type.
 55. The polypeptide of claim 53 wherein the polypeptide comprising a variant Fc region is about 2 to about 50 fold more effective in mediating ADCC relative to wild type.
 56. The polypeptide of claim 1 wherein the polypeptide comprising a variant Fc region is more effective at mediating inhibition of inflammatory immune responses relative to wild type.
 57. The polypeptide of claim 56 wherein the polypeptide comprising a variant Fc region is about 2 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type.
 58. The polypeptide of claim 56 wherein the polypeptide comprising a variant Fc region is about 10 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type.
 59. The polypeptide of claim 56 wherein the polypeptide comprising a variant Fc region is about 50 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type.
 60. The polypeptide of claim 56 wherein the polypeptide comprising a variant Fc region is about 100 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type.
 61. A method for identifying Fc variant polypeptides in silico based on calculated binding affinities to FcγRIIa, FcγRIIb and/or FcγRIIIa.
 62. The method of claim 61 comprising further calculating in silico electrostatics, solvation, packing, packing density, hydrogen binding, and entropic effects of said Fc variant polypeptides.
 63. The method of claim 61 further comprising constructing said Fc variant polypeptides and expressing said polypeptides in the context of an antibody in mammalian cells.
 64. The polypeptide of claim 1, wherein amino acid modifications do not comprise simultaneous modifications at positions 317 and 353 according to the EU index.
 65. The polypeptide of claim 1, wherein amino acid modifications do not encompass a substitution at position 332 according to the EU index. 